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Functional characterization of cytochrome b₅ reductase and its electron acceptor cytochrome b₅ in Plasmodium falciparum

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Title:
Functional characterization of cytochrome b₅ reductase and its electron acceptor cytochrome b₅ in Plasmodium falciparum
Physical Description:
Book
Language:
English
Creator:
Malvisi, Lucio
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects

Subjects / Keywords:
Plasmodium falciparum -- prevention & control   ( mesh )
Malaria, Falciparum -- drug therapy   ( mesh )
Drug Resistance   ( mesh )
Cytochrome-B(5) Reductase   ( mesh )
Cytochromes b5   ( mesh )
Artemisinins -- therapeutic use   ( mesh )
Malaria
Protein expression
Drug resistance
Arthemisinin-based combination therapy
Detoxification of xenobiotics
Dissertations, Academic -- Global Health -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Malaria is a disease of major public health importance, killing approximately one million people and causing about 250 million cases of fever annually. It mostly affects children under the age of five and pregnant women in many developing countries, making it a prominent issue in international health and maternal and child health. The most aggressive form of malaria is caused by the parasite Plasmodium falciparum which is responsible for 80% of infections and 90% of deaths from malaria, and is most prevalent in sub-Saharan Africa. Public Health interventions include the implementation of prevention programs, health education, and chemotherapy. The latter has experienced multiple problems in the past years whereby resistance of the parasite to the available drugs has emerged, rendering the majority of them ineffective.Furthermore, the high cost of those drugs represents a major obstacle to their dispensation in areas of the world where the affected people are often the less fortunate. The enzyme Cytochrome b₅ Reductase (cb₅r) and its electron acceptor Cytochrome b₅ (cb₅) play a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. Therefore, these proteins are suitable as potential novel drug targets for malaria. These two proteins have been thoroughly studied in mammals but have to be characterized in microorganisms such as fungi and parasites, including Plasmodium falciparum. It is important to note that plant cb₅r has been identified as a novel herbicidal target. Considering the close phylogenetic relationship between plant cb₅r and Plasmodium falciparum cb₅r, we conclude that these plant inhibitors may also serve as promising candidates for a new class of antimalarial drugs against the parasite.In this project, we want to obtain the biochemical and enzymatic characterization of cb₅r and cb₅ in order to establish whether these two proteins represent potential novel drug targets in Plasmodium falciparum malaria. This initial work may lead to the development of novel drugs which will consequently affect the field of public health with respect to drug delivery, drug resistance, and drug chemotherapy.
Thesis:
Thesis (M.S.P.H.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Lucio Malvisi.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 114 pages.

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aleph - 002063811
oclc - 557568083
usfldc doi - E14-SFE0003265
usfldc handle - e14.3265
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ABSTRACT: Malaria is a disease of major public health importance, killing approximately one million people and causing about 250 million cases of fever annually. It mostly affects children under the age of five and pregnant women in many developing countries, making it a prominent issue in international health and maternal and child health. The most aggressive form of malaria is caused by the parasite Plasmodium falciparum which is responsible for 80% of infections and 90% of deaths from malaria, and is most prevalent in sub-Saharan Africa. Public Health interventions include the implementation of prevention programs, health education, and chemotherapy. The latter has experienced multiple problems in the past years whereby resistance of the parasite to the available drugs has emerged, rendering the majority of them ineffective.Furthermore, the high cost of those drugs represents a major obstacle to their dispensation in areas of the world where the affected people are often the less fortunate. The enzyme Cytochrome b Reductase (cbr) and its electron acceptor Cytochrome b (cb) play a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. Therefore, these proteins are suitable as potential novel drug targets for malaria. These two proteins have been thoroughly studied in mammals but have to be characterized in microorganisms such as fungi and parasites, including Plasmodium falciparum. It is important to note that plant cbr has been identified as a novel herbicidal target. Considering the close phylogenetic relationship between plant cbr and Plasmodium falciparum cbr, we conclude that these plant inhibitors may also serve as promising candidates for a new class of antimalarial drugs against the parasite.In this project, we want to obtain the biochemical and enzymatic characterization of cbr and cb in order to establish whether these two proteins represent potential novel drug targets in Plasmodium falciparum malaria. This initial work may lead to the development of novel drugs which will consequently affect the field of public health with respect to drug delivery, drug resistance, and drug chemotherapy.
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Functional Characterization of Cytochrome b5 Reductase and its Electron Acceptor Cytochrome b5 in Plasmodium falciparum by Lucio Malvisi A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health Department of Global Health College of Public Health University of South Florida Major Professor: Wilbur Milhous, Ph.D. Andreas Seyfang, Ph.D. Azliyati Azizan, Ph.D. Date of Approval: July 27, 2009 Keywords: malaria, protein expression, drug resista nce, arthemisinin-based combination therapy, detoxification of xenobiotics Copyright 2009, Lucio Malvisi

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DEDICATION To my family and in particular to my father Martino and my mother Natalina who have supported me throughout all these years spent in th e United States, far from my home, Italy, where my heart is. I want to thank them for teaching me the important values in life that have helped me to succeed in every-day li fe, to fight in tough times and to come out of difficult situations with strength and pride I also want to thank them for the beautiful and unforgettable years I spent at home w hich made me the person I am today.

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ACKNOWLEDGEMENTS First of all I wish to thank Andreas Seyfang Ph.D., for giving the opportunity to conduct my research in his laboratory in the Department of Molecular Medicine and for providing me with everything I needed throughout the months s pent there. Secondly I want to thank Mary Jolene Holloway, Ph.D. candidate in Dr. Seyfang’s lab, and Kamisha Woolery for the time they invested in helping me ca rry out the project, their support, patience, and inspiration. Without their help this project wouldn’t have been possible. I also wish to thank my advisor Wilbur Milhous Ph.D. and Azliyati Azizan Ph.D. who were also members of my thesis committee, for they great advices in regards to this project and future steps in the development of my c areer.

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i TABLE OF CONTENTS LIST OF TABLES .................................... ................................................... ..................... iii LIST OF FIGURES ................................... ................................................... .................... iv LIST OF SYMBOLS AND ABBREVIATIONS ................. .............................................. v ABSTRACT .......................................... ................................................... .......................... vi INTRODUCTION ..................................... ................................................... ......................1 Malaria .......................................... ................................................... ........................1 History........................................... ................................................... ............1 Funding and Financial Contributions............... ............................................3 Prevention, Control, and Cure .................... .................................................6 Etiology ......................................... ................................................... ..........13 Vector and Transmission .......................... .................................................15 Life Cycle.......…………………………………………...……………….20 Public Health Impact.......………………………………..……………….21 Clinical Features ......…………………………………… ……….……….25 Pathophysiology ........…………………………………………………….27 Diagnosis.......…………………………………………………………….30 Treatment .......……………………………………………………...…….37 Treatment of Uncomplicated P. falciparum Malaria .................................40 Treatment of Malaria Caused by P. vivax P. ovale and P. malariae .......43 Malaria and Plasmodium falciparum .................................................. ...................45 Epidemiology ..................................... ................................................... .....45 Epidemiological Patterns and Transmission ......... .....................................46 Geographical Distribution ........................ ..................................................4 9 Drug Resistance .................................. ................................................... ....50 Cytochrome b5 Reductase (Cb5r) and Cytochrome b5 (Cb5) ..................................57 Cytochrome b5 Reductase ......................................... .................................57 Cytochrome b5 ................................................. ................................................... .................................. 59 Cb5r and Cb5 Function........................................... .....................................61 Cb5r and Cb5 Relationship and Interaction ..................... ...........................62 Role in Drug Metabolism and Involvement in the Deto xification of Xenobiotics through Cytochrome P450 ............... ......................................66 Phylogenetic Analysis of P. falciparum Cb5r and Cb5 ............................................ 68 Structural Comparison of P. falciparum Cb5r with Mammalian Cb5r and Other Species ............................... ...............................................70 OBJECTIVES ........................................ ................................................... .........................85 MATERIALS AND METHODS ............................. ................................................... .......88 Experimental Design .............................. ................................................... .............88

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ii Methods........................................... ................................................... ....................90 Optimization of Protein Expression ............... ................................................... .....91 Protein Purification .............................. ................................................... ...............91 RESULTS ........................................... ................................................... ............................94 Heterologous Expression of P. falciparum Cb5r Protein in E. coli .......................95 Heterologous Expression of P. falciparum Cb5 Protein in E. coli .......................102 DISCUSSION ........................................ ................................................... .......................106 REFERENCES ........................................ ................................................... .....................108

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iii LIST OF TABLES Table 1 Estimated contributions to malaria control activities by development agencies ...... ................................................... .........................4 Table 2 Funding by region (US $) .................. ................................................... .......5 Table 3 Reported malaria deaths, 1990-2007 ........ ................................................... 9 Table 4 Causes of death in children under five year s of age, estimates for 2000-2003.............. ................................................... ............................24 Table 5 Effects of some commonly used antimalarials on the infectivity of P. falciparum to the mosquito .................................................. .............40 Table 6 Levels of endemicity of malaria and associa ted statistics and characteristics ................................... ................................................... .......47 Table 7 Distribution of drug-resistant Plasmodium falciparum malaria ................51 Table 8 P. falciparum proteins with a proven role in resistance to clini cal antimalarial drugs................................. ................................................... ...56 Table 9 Kinetic properties of cb5r mutants using wild-type cb5 .......................................... 65

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iv LIST OF FIGURES Figure 1 Estimated contributions to malaria control activities by development agencies (US$) ................................................... ....................4 Figure 2 Trends in malaria cases (inpatients and outpatients) and deaths (inpatients) in rela tion to intervention, 2001 – 2006 (NMCP data) ............... ................................................... ............................. 7 Figure 3 Steps from malaria control to elimination ............... ..................................10 Figure 4a Proportion of children sleeping under ins ecticide-treated bed nets in selected countries, around 2000 and 2006 (Percentage) ................ 12 Figure 4b Number of doses of artemisinin-based comb ination therapies procured worldwide, 2003-20 06 (Millions) ..................................... ..........12 Figure 5 Global distribution of dominant or potenti ally important malaria Vectors ................... ................................................... .................................16 Figure 6 Life cycle of malaria .................... ................................................... ...........20 Figure 7 Geographical distribution of malaria ..... ................................................... .49 Figure 8 Malaria transmission areas and reported drug resist ance .......................... 52 Figure 9 Structure of rat cb5r ................................................. ...................................58 Figure 10 Cytochrome b5 diagram ........................................... ...................................60 Figure 11 Ribbon Diagram of the Docking Model of Pi g Cb5r and Cb5 .................... 64 Figure 12 Electron transport pathways to cytochrome P450 in the endoplasmic reticulum ..... ................................................... .......................67 Figure 13 Phylogram of cytochrome b5 reductases .................................................. .. 69 Figure 13a Phylogram of cytochrome b5 .................................................. ................... 70 Figure 14a Amino acid sequence alignment with the f irst two binding motifs by ClustalW analysi s and view .................................................. .... 71 Figure 14b Continued amino acid sequence alignment containing the first NADH-binding motif by Clust alW analysis and Jalview .......................... 72 Figure 14c Continued amino acid sequence alignment with the second NADH-binding motif by Clus talW analysis and Jalview ......................... 73 Figure 15 The ribbon model of rat and candida cb5r ................................................. .74

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v Figure 16 Cb5r phylogram according to the first half of the prot ein containing the FAD-binding domain and the FAD/FMN selectivity domain ........ ................................................... ...........................79 Figure 17 Cb5r phylogram according to the first half of the prot ein containing the first and se cond NADH-binding domain .......................... .84 Figure 18 Protein purification by chitin agarose ch romatography using pTWIN vector ........ ................................................... .......................93 Figure 19 DNA gel of cb5 gene coding insert by agarose gel electrophoresis ...........94 Figure 20 Optimization of P. falciparum cb5r protein expression: various IPTG concentrations .................................................. ....................98 Figure 21 P.falciparum cb5r purification gel: Chitin Agarose and Ni-NTA Chromatography ..... ................................................... ..................99 Figure 22 Optimization of P. falciparum cb5r expression gel: addition of triton and SDS detergents ........................................ ................99 Figure 23 Optimization of P. falciparum cb5r protein expression: growth at different conditions (te mperature and incubation period) .................. 100 Figure 24 Optimization of P.falciparum cb5r expression: 37C LB vs TB ............. ................................................... ........................100 Figure 25 Optimization of P. falciparum cb5r: 16C and EtOH ..............................101 Figure 26a P. falciparum cb5r protein expression screening: clones 1 through 6 ....... ................................................... .........................101 Figure 26b P. falciparum cb5r protein expression screening: clones 7 through 12 ....... ................................................... ........................102 Figure 27 Cb5 SDS-polyacrylamide gel electrophoresis ........... ...............................103 Figure 28 Cb5 gel after chitin agarose chromatography .......... .................................104 Figure 29 Cb5 gel after complete purification by Ni-NTA column chromatography ..... ................................................... ..................105

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vi LIST OF SYMBOLS AND ABBREVIATIONS Symbols and Abbreviations D escription GFATM Global Fund to fight AIDS, Tuberculosis, and Malaria PMI President’s Malaria Initiative ACT Artemisinin-based Combination Therapy INT Insecticide-treated Bed Net IRS Indoor Residual Spraying NMCP National Malaria Control Programs CDC Centers for Disease Control and Prevention WHO World Health Organization IPT Intermittent Preventive Treatment RBC Red Blood Cell RBL Reticulocyte Binding-Like DBL Duffy Binding-Like RDT Rapid Diagnostic Test PCR Polymerase Chain Reaction CQ Chloroquine CRT Chloroquine Resistance Transporter DHFR Dihydrofolate Reductase DHPS Dihydropteroate Synthetase SERCA Ca2+ Sarco/Endoplasmic Eeticulum Calcium-dependent ATPase FAD Flavin Adenine Dinucleotide NADH Nicotinamide Adenine Dinucleotide FMN Flavin Mononucleotide CBD Chitin Binding Domain

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vii TB Terrific Broth Cb5 Cytochrome b5 Cb5r Cytochrome b5 Reductase

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viii Functional Characterization of Cytochrome b5 Reductase and its Electron Acceptor Cytochrome b5 in Plasmodium falciparum Lucio Malvisi ABSTRACT Malaria is a disease of major public health importa nce, killing approximately one million people and causing about 250 million cases of fever annually. It mostly affects children under the age of five and pregnant women i n many developing countries, making it a prominent issue in international health and maternal and child health. The most aggressive form of malaria is caused by the pa rasite Plasmodium falciparum which is responsible for 80% of infections and 90% of dea ths from malaria, and is most prevalent in sub-Saharan Africa. Public Health interventions include the implementat ion of prevention programs, health education, and chemotherapy. The latter has experienced multiple problems in the past years whereby resistance of the parasite to th e available drugs has emerged, rendering the majority of them ineffective. Furthe rmore, the high cost of those drugs represents a major obstacle to their dispensation i n areas of the world where the affected people are often the less fortunate. The enzyme Cy tochrome b5 Reductase (cb5r) and its electron acceptor Cytochrome b5 (cb5) play a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxifi cation of xenobiotics. Therefore, these proteins are suitable as potential novel drug targets for malaria. These two proteins have been thoroughly studied in mammals but have to be characterized in microorganisms such as fungi and parasites, includi ng Plasmodium falciparum It is

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ix important to note that plant cb5r has been identified as a novel herbicidal target. Considering the close phylogenetic relationship bet ween plant cb5r and Plasmodium falciparum cb5r, we conclude that these plant inhibitors may also serve as promising candidates for a new class of antimalarial drugs ag ainst the parasite. In this project, we want to obtain the biochemical and enzymatic characterization of cb5r and cb5 in order to establish whether these two proteins r epresent potential novel drug targets in Plasmodium falciparum malaria. This initial work may lead to the development of no vel drugs which will consequently affect the field of public health with respect to drug delivery, drug resistance, and drug chemotherapy.

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1 INTRODUCTION Malaria Malaria is an infectious disease caused by a parasi te called Plasmodium which is transmitted via the bites of infected mosquitoes. Symptoms of malaria include fever, headache, and vomiting, and usually appear between 10 and 15 days after the mosquito bite. If not treated, malaria can quickly become l ife-threatening by disrupting the blood supply to vital organs (WHO, 2009). Each year appr oximately 500 million cases and over one million deaths occur worldwide (mainly amo ng children under five years of age), the majority being reported in Africa south o f the Sahara. These numbers make malaria one of the most important neglected disease s and a major field of action for public health organizations and authorities. A key fact is that there are incredible margins of improvement in terms of reduction of num ber of cases and deaths and this could be achieved by applying simple preventive mea sures. However, the malaria burden faces tons of issues that have impeded the regular application of those measures. Among these, the lack of funding for the development of a vaccine and of new drugs, the insurgence of drug resistance, the lack of educatio n and the cultural obstacles of the affected populations have all played an unfavorable role in the fight against malaria. All these factors are deeply related as we will discuss later. History The word malaria comes from the Italian mala aria meaning bad air, because it was associated with swamps and marshland. The dise ase was probably first described by

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2 the Chinese in 2700 BC and then again by the Greeks in the 4th century BC where it was partially responsible for the decline of their empi re. But it was again in China where a plant, named Qinghao plant, was first discovered; t his plant showed very promising antifever properties in people affected and it represen ted the only available cure for malaria for centuries. The active ingredient of Qinghao wa s isolated by Chinese scientists in 1971. Known as artemisinin, it is today a very pote nt and effective antimalarial drug, especially in combination with other medicines. An other drug that has served as one of the few known remedies to treat malaria was quinine which was derived from the bark of a plant discovered by the Spanish conquistadores in Peru. Along with artemisinin, quinine is still one of the most effective drugs av ailable today. The discovery of the parasite that causes malaria was made in 1880 by Charles Louis Alphonse Laveran, a French surgeon who notice d the presence of the parasites in the blood of a patient and for which he was awarded the Nobel Prize. Then, an Italian doctor, Camillo Golgi, found that there were differ ent types of parasites that caused malaria, one that exhibited tertian periodicity (fe ver every three days) and one that showed a quartan periodicity (fever every four days ). He also observed that the insurgence of fever corresponded to the rupture and release of merozoites (the liver stage of the parasite) into the blood. Other scientists named the various species of the parasite until the American scientist William Welch in 1897 named the malignant tertian malaria parasite, Plasmodium falciparum (CDC, 2004). Ever since, the prevalence of malari a has been steadily increasing despite the efforts of var ious public health agencies acting at local, national, and international level, to contro l and prevent the spread of the disease.

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3 Over the past twenty years the annual cases of mala ria have constantly raised and it is predicted that this trend will not slow down in the next years. The goal of reaching eradication that was proposed in the past years has appeared to be little realistic, therefore the current target is to achieve control of the dis ease. Funding and Financial Contributions Thankfully, funding for malaria that comes from int ernational organizations, federal agencies, global funds, and the governments of wealthy nations have substantially increased in the past few years (Table 1 & Fig. 1) (Roll Back Malaria, 2005). The “Global Fund to fight AIDS, Tuberculosis, and Malar ia” (GFATM), the European Union, the President’s Malaria Initiative (PMI), the “Worl d’s Bank Booster Program for Malaria Control in Africa”, and the new French-led UNITAID are some of the main contributors in the fight against malaria (European Alliance aga inst Malaria, 2007). Moreover, the intervention through personal funding by generous p eople has also occurred. These people have decided to offer their wealth in the fi ght against neglected diseases, as in the case of Bill Gates who founded, together with his w ife, “The Bill and Melinda Gates Foundation”. Its goal is to help create a more equ al world, without the burden of those infectious diseases that affect the most disadvanta geous areas of the world and the most vulnerable people (Bill and Melinda Gates Foundatio n, 2009).

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4 Table 1: Estimated contributions to malaria control activities by development agencies. (Roll Back Malaria, 2005) Figure 1: Estimated contributions to malaria contro l activities by development agencies (US$). (Roll Back Malaria, 2005) Also in the Roll Back Malaria report, we see simila r trends in the allocation of money that is made available for research in malari a. Analyzing the situation of the past few years, we notice that funds have been more and more abundant every year and in every aspect of the fight against malaria. Contrib utions from development agencies have targeted not only the governments of the countries where malaria is present, but also reached out to NGOs, research and development in ma laria control, and other organizations such as Roll Back Malaria, the Malari a Consortium, and the Special Program on Research and Training in Tropical Diseas es. Another important aspect is that funds also increased in terms of availability in di fferent geographic regions; all affected regions on earth saw their resources reach very pro mising levels. In particular sub-

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5 Saharan Africa, the area of the world where the maj ority of cases and deaths occur and where most of the fatalities are caused by the most aggressive of the malaria parasites, namely Plasmodium falciparum experienced an exponential development in the fun ds donated by agencies (Table 2) (Roll Back Malaria, 2 005). This really has a positive impact on the lives of millions of people that live in endemic areas; it is also a sign that the strategy and the administration of the budget i s well organized because funding is going where it is most needed and dispersion of mon ey, a common issue in the delivery of funds to the proper recipients, is widely avoide d. Table 2: Funding by region (US $). (Roll Back Mala ria, 2005) When looking at the data in Table 2 or many other t ables of data collected by international organizations, we realize that the hu ge difference in funding between 1999 and 2004 (the most striking numbers are observed in the sub-Saharan African region) is certainly not reflected by the crude figures of num ber of new cases and deaths. In 2005, the World Health Assembly determined to ‘ensure a r eduction in the burden of malaria by at least 50% by 2010 and by 75% by 2015’. This res olution has been interpreted to mean a reduction in morbidity as well as mortality.

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6 Prevention, Control, and Cure We are facing a worldwide burden that’s causing the death of millions of people every day and it is mainly affecting the most vulne rable part of the population, namely children and pregnant women. Current public health measures of prevention and control have served of little help in spite of their excell ent, at least theoretically, applicability. Public health authorities are acting on any possibl e form of prevention and control, attacking the problem with aggressive actions, howe ver each procedure often encounters difficulties. These can be due to drug resistance in the development of a new drug or drug target, mosquitoes that have become resistant to insecticides, or the implementation of new programs that go against cultural practices to name a few. Another very striking number that is closely associ ated to those just mentioned is the coverage of the population with curative and pr eventive measures such as Artemisinin-based Combination Therapy (ACT) and Ins ecticide-treated Bed Nets (ITN), respectively. Considering 2005 as the baseline yea r for the evaluation of the results obtained with the implementation of the new strateg ies and directives set by the WHO, such as control programs, epidemiological surveilla nce, health policies and health system structure, the target is to reach a coverage of ove r 80% of the population by 2010. It is a very complicated scenario and the data obtained fro m the surveillance reports of many countries are contradictory, often due to unreliabl e data collection. Between 2001 and 2006, many countries in Africa experienced a growth in cases and deaths even though this may be due to improved surveillance or more co mplete records for those years. On the other hand, a few other African countries such as Eritrea, Madagascar, and Rwanda, achieved high coverage of interventions and saw cas es and deaths diminished (Fig. 2).

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7 The scenario in terms of curative and preventive m easures in Africa is very variegated and difficult to interpret. But thanks to an improved epidemiological surveillance and more thorough records, more reliab le data will be available in the future that will enable us to draw more accurate conclusio ns. Figure 2: Trends in malaria cases (inpatients and o utpatients) and deaths (inpatients) in relation to intervention, 2001 – 20 06 (NMCP data). (WHO, 2008) These are two clear examples of how the start of th e implementation of malaria control programs, such as the distribution of insecticide-treated nets (ITN), indoor residual spraying (IRS), long-lasting insec ticidal nets (LLIN), and artemisinin-based combination ther apy (ACT) coincide with a stunning decrease in the number of cases and deaths. Outside Africa, the picture is similar. In a few c ountries of Central and South America, the reduction in malaria has coincided wit h improved policies on malaria control; these included efficient surveillance and early case detection to prevent and contain epidemics, integrated vector management, pr ompt diagnosis and treatment, and health system strengthening. In the WHO Eastern Me diterranean Region, the countries that have shown the greatest reductions in malaria are those where National Malaria Control Programs (NMCPs) have strong political and financial support from the government, and which operate within health systems that are well developed at the central and peripheral level. As we can see, the a vailable data do not allow us to draw definite conclusions about the efficacy of the whol e range of activities being

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8 implemented and developed by the partnership betwee n various international organizations and programs. An important aspect is that the process of reductio n of the incidence of malaria in successful countries was guaranteed by a more advan ced political and economic status. On the other hand, the absence of a firm and little corrupted head of state, the devastations caused by never-ending civil and milit ary conflicts, the simultaneous presence of other deadly diseases, a history of cen turies of subordination to colonial empire, and the scarcity of natural resources have impeded a decent administration of the malaria control interventions in certain countries such as Burkina Faso, Nigeria, Senegal, and Ghana. Here the predispositions to a well-roun ded execution of preventive and control measures are absent, therefore the logical outcome was an increase in the cases and deaths from malaria over the last few years (Ta ble 3).

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9 Table 3: Reported malaria deaths, 1990-2007. (WHO, 2008) From the table we notice the increase in deaths in the countries just mentioned: Burkina Faso, Nigeria Senegal, and Ghana where certain conditions such as lack of leadership, corruption, war, and extreme poverty prevented the malaria control programs from being successful. In an effort to a comprehensive fight against malar ia, WHO has also identified four programmatic phases on the way to achieving an d maintaining elimination: control, pre-elimination, elimination, and prevention of rei ntroduction (Fig. 3) (WHO, 2008).

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10 Figure 3: Steps from malaria control to elimination (WHO, 2008) According to this scheme, countries make the transi tion from control to the preelimination phase when less than 5% of all suspecte d malaria cases have a laboratory confirmation of malaria. The elimination phase beg ins when there is less than 1 malaria case per 1000 people at risk per year. Elimination is achieved when the “prevention of reintroduction”, without local transmission by mosq uitoes, has been successful for three or more consecutive years (WHO, 2008). Analysis of Goal 6 of the Millennium Development Go als: Combat HIV/AIDS, malaria and other diseases Interestingly, Goal 6 of the Millennium Developm ent Goals, which foresees the eradication of malaria by 2015, is fal ling short of global targets. Here, the two main means to reduce the incidence of malaria a re considered 1) prevention through a higher distribution and use of insecticide-treate d mosquito nets and 2) treatment through the use of antimalarial medicines. In part icular the United Nations are now tending to shift their focus and interest on the ar temisinin-based combination therapy, especially in sub-Saharan Africa where resistance t o traditional drugs has developed. But “There has been less progress in treating malaria t han in preventing it” UN officials say in their last report in August 2008. In fact, acco rding to data gathered from 2000 to 2006, the use of insecticide-treated mosquito nets has in creased (Fig. 4a) (even though not with

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11 the impressive figures the organization was expecti ng), while the proportion of children with fever who have received antimalarial medicines in 2006 has declined in comparison with the 2000 figures. Even more stunningly, treat ment with ACT is, in general, still at extremely low levels, even though it has shown hope ful signs of growth since 2005 (Fig. 4b). Funding for the more expensive ACT is recent but has increased markedly since 2005; moreover, the implementation of programs that try to promote the use of ACT as standard treatment for malaria is just a recent shi ft, therefore the governments and the ministries of health of the affected nations are sl owly but effectively mandating the introduction of this fairly new line of treatment. In general, evidence suggests that both the increased distribution of insecticide-treated n ets and household spraying, and the use of antimalarial medicines are giving promising resu lts in the reduction of cases and deaths from malaria around the world, even though t he idea of eradication by 2015 seems to be rather utopist (United Nations, 2008).

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12 Figure 4a: Proportion of children sleeping under in secticide-treated bed nets in selected countries, around 2000 and 2006 (Percentag e). (United Nations, 2008) Figure 4b: Number of doses of artemisinin-based com bination therapies procured worldwide, 2003-2006 (Millions). (United Nations, 2008)

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13 In 2007 the European Alliance against Malaria said that there is an estimated global requirement of about US$ 3.2 billion a year to make sure malaria is eradicated by 2015. However, at present less than US$ 1 billion per year is available for malaria resources (European Alliance against Malaria, 2007) All the elements needed for a successful eradication, such as funding, research, leadership, experience, awareness, and dedication are present but need to be used wisely. The results are promising. Etiology Malaria is caused by obligate intracellular protozo an parasites of the genus Plasmodium They are members of the class Sporozoa, therefor e alternating asexual and sexual reproductive cycles are observed. There are currently over 200 species in the genus, of which at least ten infect humans but only five are known to cause malaria in humans: Plasmodium falciparum : found in tropical regions and not established in colder climates; causes the most severe and fatal disease. Plasmodium vivax : most common and widely distributed (subtropical a nd temperate) malarial parasite; relapsing malaria. Plasmodium malariae : limited to subtropical areas. This species is le ss common than P. falciparum or P. vivax Plasmodium ovale : the least common malarial species, usually found in Africa; relapsing malaria. Plasmodium knowlesi : causes malaria in macaques but can also infect hu mans.

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14 Plasmodium falciparum has the highest rate of mortality. It is also res ponsible for 80% of malarial infections and 90% of deaths. Epis odes of infection and death from P. falciparum occur mainly in sub-Saharan Africa. P. vivax is less virulent than P. falciparum and usually not fatal. Sometimes death is observed due to splenomegaly, a pathologically enla rged spleen. Its distribution comprises Asia, Latin America, and some parts of Af rica. P. malariae causes a ‘benign malaria’ and it is not nearly as dangerous as the infections caused by P. falciparum and P.vivax. It is found worldwide from sub-Saharan Africa to Southeast Asia, the islands of the Wester n Pacific, and the Amazon Basin of South America. It is also the least studied of all five malaria-causing parasites due to its low prevalence and milder clinical manifestations. P. ovale is very limited in its distribution; it is endemic mainly in West Africa, the Philippines, Eastern Indonesia, and Papua New Guine a. It is considered less virulent than P.falciparum and P. vivax. P. knowlesi is a primate malaria parasite commonly found in So utheast Asia. As mentioned, it primarily causes malaria in macaques but it can also infect humans, especially those who work in the rain forest. With the increasing amount of deforestation in Southeast Asia, the macaques are now coming in c lose contact with humans, therefore even the people that live in semi-urban areas are n ow found to be infected with P. knowlesi. The first case of a natural infection with P.knowlesi was reported in 1965 in an American man who visited the Malaysian rainforest. P.knowlesi accounts for up to 70% of cases of malaria in Southeast Asia. This parasi te is not prevalent in West Africa. This may be due to the fact that West African population s lack the Duffy antigen, a protein on

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15 the surface of red blood cells (RBC) that the paras ite uses to start the invasion (National Institute of Health, 2000). Vector and Transmission Malaria is transmitted to humans through the bite o f infected female anopheles mosquitoes. Anopheles is the only recognized genus of mosquitoes that is able to transmit the parasite Plasmodium with the consequent development of malaria in the host. Of the 460 recognized species of anopheles, approximately 60 have been identified as vectors for malaria. Anopheles gambiae is probably the best known vector because of its ability to transmit the deadly Plasmodium falciparum and it is also the most abundant vector in Africa together with A. funestus The vector in North America is Anopheles freeborni. In India, approximately 45 mosquitoes have been ide ntified to be vectors or at least potential vectors for malaria; among these, t he most prominent are A. fluviatilis and A. minimus which are found in the foot-hill regions, A. stephensi and A. sundaicus which are numerous in the coastal regions and A. culicifacies and A. philippinensis which are characteristic of the plains (Fig. 5) (Malaria Site 2007).

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16 Figure 5: Global Distribution of Dominant or Potent ially Important Malaria Vectors. (Kiszewski et al., 2004) Understanding the biology and behavior of Anopheles mosquitoes can help understand how malaria is transmitted and can aid i n designing appropriate control strategies. One very important aspect in terms of the biology and behavior of anopheles mosquitoes is that different species have different capacities to successfully carry and transmit the parasite to humans. Some have immune mechanisms that encapsulate and kill the parasites after they have invaded the mosq uito stomach wall (Tahar et al, 2002). This ability of the mosquito immune system to ident ify and attack the parasites renders

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17 the mosquito refractory to the parasite and does no t facilitate the transmission of the disease, a fundamental characteristic in terms of p ublic health control measures. In fact, there have been studies where this immune response mechanism has been analyzed with the hope of producing genetically modified mosquito es that are refractory to malaria and that will replace the wild ones, thereby limiting t he transmission of malaria. In particular, scientists have found that the C-type lectin CEL-II I from Cucumaria echinata a sea cucumber found in the Bay of Bengal, impaired the d evelopment of the parasite Plasmodium when produced in genetically modified A. stephensi (Yoshida et al., 2007). This and further studies are setting up the base fo r the creation of transgenic mosquitoes with the goal of replacing them with the wild mosqu itoes; this would be considered a huge relief in the efforts that are being made in t he control and prevention of malaria and it is a sort of revolution in how the methods used to fight malaria are viewed. This would be considered a victory over malaria by molec ular biology rather than public health (CDC, 2008). Other behavioral characteristics that influence the transmission of malaria and that have implications on the design of control programs are 1) the sources of blood and its relation to the time required for the development o f the parasite in the mosquito, 2) the patterns of feeding, and 3) insecticide resistance. These elements are explained as follow: 1) It is known that some mosquitoes are anthropoph ilic and feed on humans while others are zoophilic and feed on animals. The two major s pecies found in Africa, A. gambiae and A. funestus are strongly anthropophilic. The next necessary process that has to take place in order for the transmission cycle to contin ue, is the development of the parasite

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18 within the mosquito. This process is known as extr insic incubation period and it ranges from 10 to 21 days, depending on the parasite speci es and the temperature at which it develops. If a mosquito does not live long enough to allow for parasite development, the transmission cannot occur. Since it is not feasibl e to measure the life span of mosquitoes in nature, researchers estimated that the rate of s urvivorship in A. gambiae in Tanzania ranged between 0.77 and 0.84, meaning that at the e nd of one day between 77% and 84% will have survived. Assuming that this survivorshi p is constant throughout the adult life of a mosquito, less than 10% of female A. gambiae would survive longer than a 14-day extrinsic incubation period (CDC, 2008). 2) Anopheles mosquitoes are crepuscular (active at dawn or dusk) or nocturnal (active at night). They enter the house between 5 p.m. and 9. 30 p.m. and again in the early hours of the morning. They start biting by late evening and the peak of biting activity is at midnight and early hours of the morning. Anopheles mosquitoes can be usually differentiated into endophagic/endophilic (mosquito es which prefer to feed indoors and rest indoors after feeding) and exophagic/exophilic (those which prefer to feed outdoors and rest outdoors after feeding); the biting patter n of the first group can be easily prevented by indoor spraying of insecticides while the activity of the second category is controlled through the destruction of the breeding sites (Malaria Site, 2007). 3) Insecticides are used in the two most efficaciou s methods of control recommended by the WHO and implemented by the ministries of health and local authorities of malariaendemic nations: indoor residual spraying and insec ticide-treated nets. Unfortunately, mosquitoes throughout the planet have shown various levels of resistance to all

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19 insecticides available. According to the CDC, ther e are over 125 mosquito species with documented resistance to one or more insecticides ( CDC, 2008). Since resistance is a fundamental aspect in control program planning, resistance management has become an integral part of resistanc e surveillance. As a consequence, program planning can also be more proficient with a n efficient surveillance. If for example, a certain vector is no longer considered s usceptible to DDT based on field surveillance and subsequent verification with molec ular tools, then an alternative insecticide may be used in that particular area. M oreover, if the vector shows some resistance to a particular insecticide, it does not mean that control activities are compromised. Resistance to a particular insecticid e must be high enough not to have any effect on preventing transmission. A good example that delineates this case scenario was Western Kenya, where resistance to pyrethroid appli ed to bed nets appeared soon after their introduction and was calculated to be approxi mately 10% of the vector population. After a couple of years, the levels of resistance w ere still the same, therefore no changes in control policies were necessary (Brogdon & McAll ister, 1998). The current available insecticides are DDT, pyreth roids (deltamethrin and permethrin), bendiocarb, and malathion. Mosquitoes have shown some degree of resistance to all of these. The most widely used i n recent years have been pyrethroids because of their low toxicity and relative safety f or humans, but resistance, particularly in the application to ITNs, has become a growing probl em throughout Africa (KerahHinzoumb et al., 2008). Furthermore, cases of cro ss-resistance to DDT and pyrethroids have been documented. This occurred because these two chemicals have a similar mode of action and act on the same target, namely the so dium channels of the nerve sheath. In

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20 order to deal with an increasing level of resistanc e to our available insecticides, it is imperative that we establish a well-organized syste m of early detection and constant field surveillance for resistance and that we make progre ss in those molecular genetics studies that will lead us to a deeper understanding of how resistance arises and maintains in populations (Brogdon & McAllister, 1998). Life Cycle Figure 6: Life Cycle of Malaria. (CDC, 2006) The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human hos t Sporozoites infect liver cells and mature into schizonts which rupture and

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21 release merozoites (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by inva ding the bloodstream weeks, or even years later). After this initial replication in th e liver (exo-erythrocytic schizogony ), the parasites undergo asexual multiplication in the ery throcytes (erythrocytic schizogony ). Merozoites infect red blood cells The ring stage trophozoites mature into schizont s, which rupture releasing merozoites Some parasites differentiate into sexual erythrocytic stages (gametocytes) Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal The parasites’ multiplication in the mosquito is known as the sporogonic cycle While in the mosquito's stomach, the microgametes penetrate the macrogametes generat ing zygotes The zygotes in turn become motile and elongated (ookinetes) which invade the midgut wall of the mosquito where they develop into oocysts The oocysts grow, rupture, and release sporozoites which make their way to the mosquito's salivary g lands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle (CDC, 2006). Public Health Impact Malaria is one of the most severe public health pr oblems worldwide and a leading cause of death and illness in many developing count ries. According to the last ‘World Malaria Report 2008’ there were an estimated 247 mi llion malaria cases among 3.3 billion people at risk in 2006, causing nearly a mi llion deaths, mostly of children under 5

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22 years. 109 countries were endemic for malaria in 20 08, of which 45 were within the WHO African Region (WHO, 2008). Other significant figures: Malaria Cases: 91% or 230 million cases were due to Plasmodium falciparum 86% of all cases occurred in the African Region, fo llowed by South-East Asia (9%) and the Eastern Mediterranean Region (3%) The percentage of cases due to P. falciparum exceed 75% in most African countries but only in a few countries outside Afric a In Africa, 19 of the most populous countries accounted for 90% of estimated cases in 2006 Malaria Deaths: There were an estimated 881.000 deaths worldwide in 2006, of which 90% in the African Region and 4% in each of the South-East Asi a and Eastern Mediterranean Region An estimated 85% of deaths occur in children under 5 years, but the proportion is much higher in the African (88%) and Eastern Medite rranean regions (76%) than in other regions (16-40%) Eighteen countries accounted for 90% of deaths in t he African Region, and seven countries had 90% of deaths outside the African Reg ion, dominated by Sudan and India So which are the most affected groups of the popula tion? For the most part children under the age of five who have not yet developed im munity to malaria, pregnant women, whose immunity is decreased by pregnancy, especiall y during the first and second

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23 pregnancies, and travelers or migrants coming from areas with little or no malaria transmission who lack immunity. A central factor that contributes to the expansion of the burden of malaria is the infection during pregnancy. Malaria can have adver se effects on both the mother and the fetus. Health consequences include maternal anemia fetal loss, premature delivery, intrauterine growth retardation, and delivery of lo w birth-weight infants (<2500 g or <5.5 pounds). The health condition that an infection ca uses differ according to the type of malaria transmission area. There are two types of transmission areas: stable (high) or unstable (low). In high transmission areas, women have gained a l evel of immunity to malaria that wanes somewhat during pregnancy. Malaria infe ction is more likely to result in severe maternal anemia and delivery of low birth -weight infants. In low transmission areas, women generally have d eveloped no immunity to malaria. Malaria infection is more likely to resul t in severe malaria disease, maternal anemia, premature delivery, or fetal loss. In sub-Saharan Africa, the region of the world with the highest incidence of malaria, infections are estimated to cause 400,000 cases of severe maternal anemia and 75,000-200,000 infant deaths annually. Maternal an emia contributes significantly to maternal mortality and causes an estimated 10,000 d eaths per year. Low birth weight is the greatest risk factor for neonatal mortality and a major contributor to infant mortality. Although many factors contribute to low birth weigh t, malaria is a major factor and one

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24 of the few, along with poor nutrition, anemia, and other infections, that is amenable to intervention once a woman becomes pregnant (CDC, 20 04; Steketee et al., 2001). In an effort to combat this problem, the World Hea lth Organization currently recommends a three-pronged approach to prevent thes e adverse effects in areas of Africa with high levels of transmission of Plasmodium falciparum malaria: 1. Intermittent preventive treatment (IPT) with antima larial drugs 2. Insecticide-treated bed nets (ITN) 3. Febrile malaria case management One very important point that’s worth to be mention ed here is that the available health information for much of sub-Saharan Africa i s of very poor quality. The annual estimates provide a useful tool that greatly aids i n the types, number, and extensiveness of malaria control initiatives but there has been s kepticism about their origin and validity. Therefore, all present approximations are derived f rom evidence-based approaches that provide greater credibility to malaria control proj ects (Snow et al., 1999). Often times e stimates of the number of malaria deaths are made b y: (1) multiplying the estimated number of P. falciparum malaria cases by a fixed case-fatality rate for ea ch country; or (2) from an empirical relationship between measures of malaria transmission risk and malaria-specific mortality rates (WHO, 2008). Rank Cause Numbers (thousands per year) % of all deaths 1 Neonatal causes 3,910 37 2 Acute respiratory infections 2,027 19 3 Diarrheal diseases 1,762 17 4 Malaria 853 8

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25 5 Measles 395 4 6 HIV/AIDS 321 3 7 Injuries 305 3 Other causes 1,022 10 Total 10,596 100.0 Table 4: Leading Causes of Death in Children Under Five Years of Age, Estimates for 2000-2003. (WHO, 2005) Clinical Features Infection with malaria parasites may result in a w ide variety of symptoms, ranging from absent or very mild symptoms to severe disease and even death (CDC, 2006). In general, malaria presents itself as a fe brile illness characterized by fever and related symptoms (Malaria Site, 2006). The incubat ion period can range from 7 to 30 days, the shortest time being typically associated with P. falciparum and the longest interval with P. malariae In the case where antimalarial drugs are being t aken for prophylaxis and in the event of an infection with P. vivax or P. ovale the incubation period can even reach months or years because these two parasites can produce dormant liver stages and reactivate after months or even ye ars after the first manifestation, a physiological process known as relapse. Malaria can be categorized as uncomplicated and se vere (complicated). In the uncomplicated malaria a typical attack lasts about 6-10 hours. It consists of: a cold stage (sensation of cold, shivering) a hot stage (fever, headaches, vomiting; seizures i n young children) a sweating stage (sweats, return to normal temperat ure, tiredness).

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26 Attacks occur every second day with tertian parasit es ( P. falciparum P. vivax and P. ovale ), every third day with the quartan parasite ( P. malariae ), whereas a quotidian fever (every day) is observed in P. knowlesi Additional clinical manifestations of P. falciparum include mild jaundice, enlargement of the liver, a nd increased respiratory rate. In countries where cases of mala ria are infrequent, the typical symptoms may be attributed to influenza, a cold, or other co mmon infections, especially if malaria is not suspected. Conversely, in countries where m alaria is frequent, residents often recognize the symptoms as malaria and treat themsel ves without seeking diagnostic confirmation ("presumptive treatment"). Severe malaria occurs when P. falciparum infections are complicated by serious organ failures or abnormalities in the patient's bl ood or metabolism. The manifestations of severe malaria include: Cerebral malaria, abnormal behavior, impairment of consciousness, seizures, coma, or other neurologic abnormalities Severe anemia due to hemolysis (destruction of the red blood cells) Hemoglobinuria (hemoglobin in the urine) due to hem olysis Pulmonary edema (fluid buildup in the lungs) or acu te respiratory distress syndrome (ARDS), which may occur even after the par asite counts have decreased in response to treatment Abnormalities in blood coagulation and thrombocytop enia (decrease in blood platelets) Cardiovascular collapse and shock

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27 Severe malaria occurs most often in persons who hav e no immunity to malaria or whose immunity has decreased. These include all re sidents of areas with low or no malaria transmission, and young children and pregna nt women in areas with high transmission (CDC, 2006). Pathophysiology An important and fairly new concept in describing malaria is that of a multisystem disorder even when the clinical manifes tations appear to involve a single organ, such as the brain or the liver. For instanc e, metabolic acidosis that is often observed in severe cases of malaria, such as cerebr al malaria or malarial anemia, may lead to a syndrome of respiratory distress (Taylor et al., 1993). In most cases, this is predominantly (but not exclusively) a lactic acidos is (English et al., 1996). To emphasize the complexity, variety, and complementary nature o f clinical and molecular events of malaria, it was determined that lactic acidosis in children has multiple causes: from increased production by parasites, through direct s timulation by cytokines, to decreased clearance by the liver. However, most significantl y by far is likely to be the combined effects of several factors in reducing oxygen deliv ery to tissues (English et al., 1997). A second characteristic is that there is no simple one-to-one correlation between clinical syndromes of severe disease and pathogenic processes. As a result, severe anemia may arise from multiple poorly understood pr ocesses, including acute hemolysis of uninfected RBCs and dyserythropoiesis, as well a s through the interaction of malaria infection with other parasite infections and with n utritional deficiencies (Newton et al., 1997).

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28 But how does the infection begin at the physiologi cal level? Mosquitoes inject parasites (sporozoites) into the subcutaneous tissu e and less frequently directly into the bloodstream; from there, sporozoites travel to the liver. Recent evidence indicates that sporozoites pass through several hepatocytes before invasion is followed by parasite development (Mota et al., 2001). The co-receptor o n sporozoites for invasion involves, in part, the thrombospondin domains on the circumsp orozoite protein and on thrombospondin-related adhesive protein. These dom ains bind specifically to heparin sulfate proteoglycans on hepatocytes in the region in apposition to sinusoidal endothelium and Kuppfer cells (Frevert et al., 1993 ). Within the hepatocyte, each sporozoite develops into tens of thousands of meroz oites, each able to invade a RBC upon release from the liver. Disease begins only o nce the asexual parasite multiplies within RBCs. This is the only gateway to disease. P. falciparum and P. vivax within RBCs develop over 48 hours, producing around 20 mer ozoites in a mature parasite, each able to invade other RBCs. A small proportion of a sexual parasites convert to gametocytes that are critical for the transmission of the infection to other hosts through female anopheline mosquitoes. This does not cause any disease. In this context, the strategy of P. vivax differs from that of P. falciparum P. vivax develops into gametocytes soon after release of mer ozoites from the liver while P. falciparum gametocytes develop much later. Early treatment of clinical malaria by antibloodstage chemotherapy for P. falciparum also kills the developing gametocytes; P. vivax transmits before the symptomatic stage of the disea se (Weatherall et al., 2002). All the typical clinical symptomology and severe di sease pathology associated with malaria is caused by the asexual erythrocytic or blood stage parasites (CDC, 2004).

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29 The sequence of invasion into the red blood cells i s probably similar for all Plasmodium species. The merozoite first attaches to red cells In P. falciparum Erythrocyte Binding Antigen 175 and Merozoite Surface Protein 1, 2 with sialoglycoproteins have been identified as the ligands while in P. vivax the Duffy antigen on RBC is the site of binding. After the attachment to the red cell, the merozoite re-orientates itself so that apposition of apical end occurs. This is followed by localized invagination and interiorization of the merozoite. The entire proce ss is completed in 30 seconds. The growth and multiplication cycle within the RBCs ( Erythrocytic schizogony ) takes about 48 hours for one cycle (72 hours in case of P. malariae ). Each merozoite divides into 8-32 (average 10) fresh merozoites. The merozoites grow in stages into rings called trophozoites (trophos means nourish) and divide in a Schizont meaning split to release more merozoites (mero means separate). At the end of this cycle, the mature schizonts rupture the RBCs and release the new merozoites int o the blood, which in turn infect more RBCs. The merozoite ingests hemoglobin from R BCs to form a food vacuole where it is degraded and heme is released. The tox ic heme is in turn detoxified by heme polymerase and sequestrated as hemozoin. All the c linical features of malaria are caused by these events in the blood. Eventually, the grow ing parasite progressively consumes and degrades intracellular proteins, principally he moglobin, resulting in the formation of the 'malarial pigment' and hemolysis of the infecte d red cell. This also alters the transport properties of the red cell membrane, and the red ce ll becomes more spherical and less deformable. The rupture of red blood cells by mero zoites releases certain factors and toxins, which could directly induce the release of cytokines such as TNF and interleukin-

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30 1 from macrophages, resulting in chills and high gr ade fever. This occurs once in 48 hours, corresponding to the erythrocytic cycle (Wea therall et al., 2002). Both P. falciparum and P. vivax can cause severe anemia, but only P. falciparum causes the multiple complications of cerebral malar ia, hypoglycemia, metabolic acidosis, and respiratory distress. Certain differences in t he biology of the two parasites partially explain the differences in patterns of disease. Fi rst, P. falciparum can invade a large percentage of the RBCs, whereas P. vivax is limited to reticulocytes. A second difference between the two parasites is that there is a surpri sing redundancy of invasion pathways in P. falciparum P. vivax invades only Duffy blood group-positive RBC23 and i s largely limited to reticulocytes. In West Africa, where th e Duffy blood group is missing on RBCs, P. vivax essentially disappeared. The limitations in invas ion of P. vivax have led to the discovery of two families of parasite recept ors: 1) the parasite molecule that binds to the Duffy blood group system and Duffy binding-l ike (DBL) homologous proteins of P. falciparum and P. knowlesi (Kappe et al., 1998) and 2) the parasite reticulocytebinding proteins of P. vivax (Galinski et al., 1992) and reticulocyte binding-l ike (RBL) homologous proteins of P. falciparum (Rayner et al., 2001). The various members of the DBL and RBL families may recognize different RBC re ceptors than the Duffy blood group or the receptor on reticulocytes. Diagnosis The diagnosis of malaria involves identification of malaria parasite or its antigens/products in the blood of the patient. Alt hough this seems simple, the efficacy of the diagnosis is subject to many factors. The diff erent forms of the four malaria species,

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31 the different stages of erythrocytic schizogony, th e endemicity of different species, the population movements, the interrelation between the levels of transmission, immunity, parasitemia, and symptoms, the problems of recurren t malaria, drug resistance, persisting viable or non-viable parasitemia, and sequestration of the parasites in the deeper tissues all affect proper diagnosis. Finally the use of ch emoprophylaxis or even presumptive treatment on the basis of clinical diagnosis can al l have a bearing on the identification and interpretation of malaria parasitemia on a diag nostic test (Malaria Site, 2006). Prompt and accurate diagnosis is the key to effecti ve disease management, which is one of the main interventions of the Global Mala ria Control Strategy (WHO, 1993). It is thus of concern that poor diagnosis continues to hinder effective malaria control. This is due to a combination of factors, including non-s pecific clinical presentation of the disease, high prevalence of asymptomatic infection in some areas, lack of resources and insufficient access to trained health care provider s and health facilities, and widespread practice of self-treatment for clinically suspected malaria. The first and most practical method for diagnosing malaria is through clinical examination. Clinical diagnosis is the most widely used approach in rural areas and at the periphery of the health care system where labor atory support to clinical diagnosis does not exist (WHO/USAID, 2000). The problem with clinical diagnosis is that the symptoms of malaria are very non-specific, especial ly those of mild and moderate malaria. A diagnosis of malaria based on clinical grounds alone is therefore unreliable. As a result, when malaria is suspected, the clinica l examination should be followed by a microscopic diagnosis, which represents the establi shed method for laboratory confirmation of malaria. In most settings, the proc edure consists of collecting a finger-

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32 prick blood sample, preparing a thick blood smear ( in some settings a thin smear is also prepared), staining the smear (most frequently with Giemsa), and examining the smear through a microscope (preferably with a 100X oil-im mersion objective) for the presence of malaria parasites (Makler et al., 1998). Microscopy offers many advantages. It is sensitive. When used by skilled and careful technicians, microscopy can detect densities as low as 5 – 10 parasites per l of blood (WHO, 1990). Under general field conditions, however, the detection ca pabilities of a typical microscopist might be more realistically placed at 100 parasites per l of blood (WHO, 1998). It is informative. When parasites are found, they can be characterized in terms of their species ( P. falciparum P. vivax P. ovale and/or P. malariae ) and of the circulating stage (e.g. trophozoites, schizonts, ga metocytes). Occasionally, expert microscopists can detect morphological alterations induced by recent drug treatment. In addition, the parasite densities can be quantified (from ratio of parasites per number of leukocytes or erythrocytes) Such quantifications are needed to demonstrate hyperparasitemia (which may b e associated with severe malaria) or to assess parasitological response to c hemotherapy. It is relatively inexpensive. Cost estimates for e ndemic countries range from about US$ 0.12 to US$ 0.40 per slide examined (Palm er, 1999). Such figures, however, do not reflect the true cost to the health system or to the patient, which may be substantially higher. In addition, the cost per test will increase if

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33 utilization is low, or if microscopy in the health facility is used only for malaria diagnosis. It is a general diagnostic technique that can be sh ared with other disease control programs, such as those against tuberculosis or sex ually transmitted diseases. It can provide a permanent record (the smears) of t he diagnostic findings and be subject to quality control. Microscopy suffers from three main disadvantages. It is labor-intensive and time-consuming, normally requiring at least 60 minutes from specimen collection to result. It is exacting and depends absolutely on good techn iques, reagents, microscopes and, most importantly, well trained and well superv ised technicians. Unfortunately these conditions are often not met, p articularly at the more peripheral levels of the health care system. There are often long delays in providing the micros copy results to the clinician, so that decisions on treatment are often taken without the benefit of the results. There are also non-microscopic tests that can be gr ouped in a category named ‘Rapid Diagnostic Tests’ (RDT). These tests involve ident ification of the parasitic antigen or the anti-plasmodial antibodies, or the parasitic metabo lic products. Some of the most common RTDs are: OptiMal Assay, Polymerase Chain Re action (PCR), the immunechromatographic test, the detection of antibodies b y Radio-immuno assay, and immunofluorescence or enzyme immune assay.

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34 The advantages of RDTs over microscopy are: RDTs are simpler to perform and to interpret. They do not require electricity, special equipment or training in microscopy. Perip heral health workers (and other health providers as well as community volunteers) c an be taught the procedure in a matter of hours, with good retention of skills ov er a one-year period. RDTs are relatively robust and test performance and interpretation vary relatively little among individual users. Moreover, most kits can be shipped and stored under ambient conditions. Since RDTs detect circulating antigens, they may de tect P. falciparum infection even when the parasites are sequestered in the deep vascular compartment and thus undetectable by microscopic examination of a p eripheral blood smear. In women with placental malaria (as demonstrated by pl acental smears), RDTs have detected circulating HRP-II even though the blood s mears were negative due to sequestration of P. falciparum in the placenta (Leke et al., 1999). Disadvantages include: Commercially available RDTs targeting HRP-II can de tect only P. falciparum Such kits will detect only a portion of cases in ar eas where other Plasmodium species are co-endemic. They are not suitable for diagnosing cases of imported malaria from areas where P. falciparum is not necessarily the most prevalent species. RDTs that target HRP-II of P. falciparum can give positive results for up to two weeks following chemotherapy and parasite clearance as confirmed by

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35 microscopy. The reason for this antigen persistenc e needs to be clarified. Pending such clarification, RDTs targeting HRP-II might yie ld confusing results in relation to the assessment of treatment failure or drug resi stance. The current RDTs are more expensive than microscopy with costs per test varying from US$ 0.60 to US$ 2.50 and possibly more depending on the marketing area. RDTs are not quantitative. They thus fail to provi de information of possible prognostic importance and are not suitable for deta iled investigations on the therapeutic efficacy of antimalarial drugs. Kits that detect both P. facilparum and non-falciparum species cannot differentiate between P. vivax P. ovale and P. malariae nor can they distinguish pure P. falciparum infections from mixed infections that include P. falciparum RDTs that detect antigens produced by gametocytes c an give positive results in infections where only gametocytes are present. Gam etocytes are not pathogenic, and gametocytes of P. falciparum can persist following chemotherapy without implying drug resistance. Such positive RDT result s can lead to erroneous interpretations (false positives) and unnecessary t reatment of people not suffering from malaria. Now a differentiation has to be made between the di agnostic tools of choice in areas of high and low malaria transmission. In high malaria endemicity areas, if a case is suspected of severe malaria, laboratory confirmatio n can guide initial therapy. In facilities at the central and district levels, micr oscopy should be the confirmatory diagnostic test of choice. In peripheral locations where microscopy is not available,

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36 RDTs might prove particularly useful since they can be performed by health workers with limited training and skills. Compared to bloo d smears, RDTs provide more timely results for disease management. In low malaria end emicity areas, clinical diagnosis should be rapidly followed by microscopy and RDTs i f necessary. In the extreme case of remote communities or highly mobile populations, wh ere microscopic diagnosis is not available and where patients do not have adequate a ccess to health care facilities, treatment is frequently based on clinical diagnosis alone. Here, the use of RDTs by local health workers or community volunteers has proved v aluable. Another important factor to consider is the develop ment of drug resistance. In fact, when the diagnosis of malaria is based merely on a symptomatic evaluation, many problems can emerge. First of all, since malaria h as very unspecific symptoms, a diagnosis can never be accurate, and using malarial medicines when the agent causing the disease is not a parasite in the genus Plasmodium greatly enhances the development of resistance. Secondly, resistance to available drug s in many areas of the world is an emerging problem, it is species specific and it has a geographical distribution; therefore prescribing a certain medicine ignoring what Plasmodium species is causing the infection, can strengthen their resistance and expa nd the problem. A series of wrong diagnosis has certainly played a role in the develo pment of drug resistance worldwide, especially to the two traditional medicines that ha ve represented firstor second-line treatment for many decades: chloroquine and sulfado xine–pyrimethamine. This has lead to the need of developing new antimalarial drugs; t oday public health authorities, in order to avoid an increase in the resistance to the tradi tional medicines, recommend the use of a fairly new group of drugs to which all Plasmodium species have shown susceptibility:

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37 artemisinin and its derivatives. The downside of a rtemisinin is its relatively high cost and lower safety. Treatment The development of resistance to the available drug s in the last few decades has posed a serious challenge in our goal of achieving malaria eradication and has evolved our way of facing and managing the disease. It is undoubtedly a complicated problem. Until a few decades ago, chloroquine was a very eff ective medicine and no signs of resistance had yet appeared. Then, the indiscrimin ate use of chloroquine posed a very heavy selective pressure on the parasite and this p rocess slowly lead to the insurgence of resistant strains that survived the action of the d rug. As time went by, resistant strains were effectively reproducing and allowed for a succ essful transmission; this created a worldwide resistance of P. falciparum to most of the available medicines, such as amodiaquine, chloroquine, mefloquine, quinine, and sulfadoxine-pyrimethamine. As we know, P. falciparum is the most prevalent and dangerous malarial paras ite, hence, in this discussion, we will mainly focus on the treatment o f this form of malign malaria. There is a series of factors that allowed the paras ite to develop resistance. Some of these factors are: the extremely high prevalence of the disease throughout the world, the administration of drugs when malaria was only s uspected through clinical symptoms (and the patient was malaria negative), the poor ad herence to the recommended therapy, the prescription of ineffective drugs against a spe cific species of parasite, and the use of monotherapy (single drug against the parasite) as o pposed to combination therapy, in

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38 which two different drugs have two distinct modes of action on the parasite (Hastings & Mackinnon, 1998). Resistance to antimalarials has been documented for P. falciparum P. vivax and, recently, P. malariae In P. falciparum resistance has been observed to almost all currently used antimalarials (amodiaquine, chloroqu ine, mefloquine, quinine and sulfadoxine–pyrimethamine) except for artemisinin a nd its derivatives. The geographical distribution and rates of diffusion have varied con siderably. P. vivax has rapidly developed resistance to sulfadoxine–pyrimethamine i n many areas. Chloroquine resistance is confined largely to Indonesia, East T imor, Papua New Guinea and other parts of Oceania. There are also documented report s from Peru. P. vivax remains sensitive to chloroquine in South-East Asia, the In dian subcontinent, the Korean peninsula, the Middle East, North-East Africa, and most of South and Central America (WHO, 2006). As it can easily be extrapolated, treatment of mala ria today is a very complex topic. First, a number of general properties of th e current antimalarial drugs will be delineated; this will be followed by an in-depth di scussion of the specific medicines against different parasites and their different typ es of combination, settings, and interventions. Generally speaking, antimalarials c an help bring about a reduction in malaria transmission by their effect on parasite in fectivity. This can be a direct effect on the gametocytes, the infective stages found in huma n infections (gametocytocidal effect) or, when the drug is taken up in the blood meal of the mosquito, an effect on the parasite’s development in the insect (sporonticidal effect). Chloroquine (CQ) acts against young gametocytes but has no suppressive effects on mature infective forms (Bruce-

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39 Chwatt, 1981). Chloroquine has even been shown to be capable of enhancing the infectivity of gametocytes to the mosquito (Hogh et al., 1998). In contrast, sulfadoxinepyrimethamine (SP) increases gametocyte carriage bu t, provided there is no resistance, reduces the infectivity of gametocytes to mosquitoe s (Hogh et al., 1998; Robert et al., 2000; von Seidlein et al., 2001). Artemisinins are the most potent gametocytocidal drugs among those currently being used to treat an asexua l blood infection (von Seidlein et al., 1998; Targett et al., 2001; Drakeley et al., 2004). They destroy immature gametocytes, preventing new infective gametocytes from entering the circulation, but their effects on mature gametocytes are less, hence they will not af fect the infectivity of those already present in the circulation at the time a patient pr esents for treatment (Pukrittayakamee et al 2004). Primaquine, an 8-aminoquinoline antimalar ial that has been widely used as a hypnozoiticidal drug, is the only antimalarial medi cine that had been deployed in the treatment of P. falciparum infections due to its effects on infectivity. It a cts on mature infective gametocytes in the circulation and accele rates gametocyte clearance (Pukrittayakamee et al 2004), as opposed to artemisinins, which mainly i nhibit gametocyte development. Amodiaquine is an antimalarial with schizonticidal activity. It is effective against the erythrocytic stages of all four species of Plasmodium falciparum It is as effective as chloroquine against chloroqui ne-sensitive strains of Plasmodium falciparum and it is also effective against some chloroquine-r esistant strains. Amodiaquine accumulates in the lysosomes and brings about loss of function. The parasite is unable to digest hemoglobin on which it depends for its energy (‘Crusade Against Malaria’, 2008). Lastly, mefloquine is ind icated for the treatment of mild to moderate acute malaria caused by mefloquine-suscept ible strains of P. falciparum (both

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40 chloroquine-susceptible and resistant strains) or b y Plasmodium vivax. There are insufficient clinical data to document the effect o f mefloquine in malaria caused by P. ovale or P. malariae. Patients with acute P. vivax malaria, treated with mefloquine, are at high risk of relapse because mefloquine does not el iminate exoerythrocytic (hepatic phase) parasites (RxList, 2008). Table 5: Effects of Some Commonly Used Antimalarial s on the Infectivity of P. falciparum to the Mosquito. (WHO, 2006) Treatment of Uncomplicated P. falciparum Malaria Today the World Health Organization recommends com bination therapy for the treatment of falciparum malaria. Monotherapy is st rongly discouraged as either resistance is already present or it may lead to the process of resistance development. The parasites that survive have genes that make them re sistant to a certain drug and these

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41 same genes can then be transmitted to the mosquito when it bites and, consequently, to humans (Hallett et al., 2004). Antimalarial combination therapy is the simultaneou s use of two or more blood schizontocidal drugs with independent modes of acti on and thus unrelated biochemical targets in the parasite. The concept is based on t he potential of two or more simultaneously administered schizontocidal drugs wi th independent modes of action to improve therapeutic efficacy and also to delay the development of resistance to the individual components of the combination (WHO, 2006 ). The recommended treatment for the cure of falciparu m malaria is the Artemisinin-based Combination Therapy (ACT). There are four derivatives of artemisinin that induce a rapid clearance of parasi temia and a resolution of the symptoms. They reduce the parasite number by a factor of appr oximately 10000 in each asexual cycle, which is more than other current antimalaria ls (100to 1000-fold per cycle). Artemisinin and its derivatives are also rapidly el iminated. When given in combination with rapidly eliminated compounds (tetracyclines, c lindamycin), a 7-day course of treatment with an artemisinin compound is required; but when given in combination with slowly eliminated antimalarials, shorter courses of treatment (3 days) are effective. A trial that compared the effects of a single drug su ch as amodiaquine, mefloquine, or sulfadoxine-pyrimethamine with that of one of those same drugs with the addition of artesunate (an artemisinin derivative), found that there was a clear benefit in the use of the combination treatment. It also resulted in few er parasitological failures at day 28 which is the current recommended duration of follow -up. This is when the treatment is considered to be successful, even though recrudesce nce may occur and another cycle

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42 needs to be begun with the same firs-line treatment or, if the circumstances suggest that sensitivity is not sufficient, then the second-line treatment may be used. Another characteristic that make artemisinin compounds idea l for the treatment of malaria is that they are active against all four species of malaria parasites and are generally well tolerated. As previously mentioned, these drugs al so have the advantage, from a public health perspective, ofreducing gametocyte carriage and thus the transmiss ibility of malaria. This contributes to malaria control in ar eas of low endemicity (WHO, 2006). As mentioned, the concept of ACT is based on the us e of an artemisinin derivative and the choice of another drug. Whereas no significant differences in the efficacy of the artemisinin product in terms of abs orption and bioavailability have been demonstrated, it is the properties of the partner m edicine that determines the effectiveness and choice of combination. Various combination the rapies have been evaluated in multiple clinical trials where an artemisinin compo und was given in conjunction with another drug such as amodiaquine, chloroquine, lume fantrine, mefloquine, piperaquine, and doxycycline. The combinations that proved to b e the most advantageous were (in alphabetical order): artemether-lumefantrine artesunate + amodiaquine artesunate + mefloquine artesunate + sulfadoxine-pyrimethamine Although for many countries, artemether-lumefantrin e and artesunate + mefloquine may give the highest cure rates, there m ay be problems of affordability and availability of these products. Also, there is cur rently insufficient safety and tolerability

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43 data on artesunate + mefloquine at the recommended dose of 25mg/kg in African children to support its recommendation there. Tria ls with mefloquine monotherapy (25mg/kg) have raised concerns of tolerability in A frican children. Countries may therefore opt to use artesunate + amodiaquine and a rtesunate + sulfadoxine– pyrimethamine, although they may have lower cure ra tes because of resistance (WHO, 2006). Finally, a last note on a crucial aspect of malari a treatment: treatment management. In order to optimize the benefit of dep loying ACTs, and to have an impact on malaria, it will be necessary to deploy them as widely as possible; this means that they will have to reach the most peripheral health cente rs and communities. The dissemination of clear national treatment guideline s, use of appropriate information, education, communication materials, monitoring of t he deployment process, access and coverage, and the provision of adequately packaged antimalarials are needed to optimize the benefits of providing these new effective treat ments (WHO, 2006). Treatment of Malaria Caused by P. vivax, P. ovale, and P. malariae P. vivax has been characterized thoroughly in terms of phar maceutical sensitivity and resistance due to its high prevalence in endemi c areas of the Middle East, Oceania, and Central and South America. In Africa, it is ra re except in the Horn and it is almost absent in West Africa. In most areas where P. vivax is prevalent, malaria transmission rates are low, and the affected populations therefo re achieve little immunity to this parasite. Consequently, people of all ages are at risk.

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44 The other two human malaria parasite species, P. malariae and P. ovale, are generally less prevalent but are distributed worldw ide, especially in the tropical areas of Africa. Of these three parasites, only P. vivax and P. ovale form hypnozoites, the liver stage of the parasite that can result in relapses o f infection. This affects the development and schooling of children and debilitates adults, t hereby impairing human and economic development in affected populations. Therefore, wh en treating these two forms of malaria, the goal is to cure both the blood stage a nd the liver stage infections, thereby preventing both relapses and recrudescence. This i s known as radical cure. Recent data on the in vivo susceptibility of P. ovale and P. malariae are very few. Both are regarded as very sensitive to chloroquine, although there is a single recent report of chloroquine resistance in P. malariae They are also thought to be susceptible to amodiaquine, mefloquine, and the artemisinin produc ts. On the other hand, P. vivax has received much more attention and therefore we posse ss very extensive studies that assess the susceptibility to drugs of this parasite. It i s still generally very sensitive to chloroquine, although resistance has emerged in som e areas such as Oceania, Indonesia, and Peru. Resistance to pyrimethamine has increased rapidly in some areas, and sulfadoxine-pyrimethamine is consequently ineffecti ve. In general, P. vivax is sensitive to all the other antimalarial drugs; it is more sen sitive than P. falciparum to the artemisinin derivatives, and slightly less sensitiv e to mefloquine (although mefloquine is still effective). The only drugs with significant activity against the hypnozoites are the 8aminoquinolines.

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45 As far as P. ovale is concerned, the recommended treatment for the re lapsing malaria is the same as that given for radical cure in P. vivax i.e. with chloroquine and primaquine. Instead, P. malariae should be treated with the standard regimen of chloroquine as for vivax malaria, but it does not r equire radical cure with primaquine as no hypnozoites are formed in infection with this sp ecies. Malaria and Plasmodium falciparum Epidemiology First of all, it is important to consider that ther e are three main factors that determine the occurrence of malaria. These factors also corr espond to the three components of the malaria cycle: 1. In order for the parasite to complete the "inverteb rate host" half of their life cycle, Anopheles mosquitoes must be present and must be in contact with humans. 2. In order for the parasites to complete the "vertebr ate host" half of their life cycle, humans must be present and must be in contact with Anopheles mosquitoes. 3. Malaria parasites must be present (CDC, 2004). Climate can also influence all three components of the life cycle. It is thus a key determinant in the geographic distribution and the seasonality of malaria. An essential resource for the successfull reproduction and devel opment of the mosquito eggs is the presence of water. The required time for the eggs to develop into larvae, pupae, and adults, is approximately 9-12 days in tropical area s. In case rain is scarce and water dries

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46 up too quickly, the egg is not able to complete its evolution; on the other hand, when rains are too excessive, eggs are can be damaged an d flushed away. Once adult mosquitoes have emerged, the ambient tem perature, humidity, and rains will determine their chances of survival. On ce an adult mosquito takes a bite on an infected human, the parasite needs to go through a whole new growth cycle inside the mosquito, known as the extrinsic cycle. This cycle takes about 9 to 21 days to complete at 25C, meaning that the mosquito needs to survive long enough to allow the extrinsic cycle to complete. Warmer climates shorten the ext rinsic cycle, thus increasing the chances of transmission. Conversely, below a certa in temperature (15C for Plasmodium vivax 20C for P. falciparum ), the extrinsic cycle cannot be completed and mala ria cannot be transmitted (Craig et al., 1999). Epidemiological Patterns, Transmission, and Distrib ution There is a fundamental concept that describes and i nfluences the epidemiology and distribution of malaria: transmission. Transmi ssion is described in terms of intensity, stability, and seasonal patterns. The intensity of transmission can be measured as the average number of infectious bites received during a given period of time by an individual living in the area.Stability regards the situation over a period of ye ars. In areas of stable transmission, the pattern of transm ission remains roughly unchanged from year to year, whereas areas with unstable malaria a re characterized by considerable variation in the intensity of transmission between years. In areas of stable malaria transmission, the transmission intensity is roughly reflected in the spleen rates and the point prevalence of parasitemia in children (Gilles 1993).

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47 According to the levels of spleen rate and parasite mia, endemicity is comprised of four categories: holoendemic, hyperendemic, mesoend emic, and hypoendemic. In holoendemic area, transmission occurs all year long In hyperendemic areas, transmission is intense but with periods of no tran smission during the dry season. In mesoendemic regions, there is a regular seasonal tr ansmission. Finally, in hypoendemic areas, transmission is very intermittent (Shiff, 20 06). Table 6 summarizes the characteristics of the four different levels of end emicity. Spleen rate*in < 5 year old Parasitemia <5 year old Transmission Clinical Manifestations Mortality Holoendemic > 75% 60 – 70 % Stable** Anemia severe in early life High in 1st and 2nd year of life Hyperendemic 50 – 70 % 50 – 70 % Seasonal but intense. Stable Cerebral malaria in older children Mesoendemic 20 – 50 % < 20% Seasonal. Unstable*** Cerebral malaria common Hypoendemic 0 – 10 % 0 – 10 % Periodic/Unusu al rainfall. Unstable Severe outbreaks in children/adults High in all groups and ages Table 6: Levels of Endemicity of Malaria and Associ ated Statistics and Characteristics. (Shiff, 2006) Spleen Rate: number of palpable enlarged spleens per 100 individuals of similar ages. ** Stable malaria occurs in areas of high prevalenc e, cases are frequently asymptomatic, fever occurence is infrequent, anemia is high at yo unger ages (particularly under 2 years of age), and mortality is greatest under 2 years of age (Shiff, 2006). *** Unstable malaria is based on immunity achieved through personal exposure to Plasmodium antigens. Since exposure in areas of low endemici ty is usually low, the immune system is naive to the antigens and is not a ble to effectively produce a strong

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48 immune response; a broad range of disease situation s is possible, including cerebral malaria in children 4 – 6 years old and adult malar ia. Since the infection is highly symptomatic, a rapid diagnosis is critical and prom pt effective treatment is vital to avoid fatalities (Shiff, 2006). Now why do children over five years of age and adul ts living in areas of stable malaria transmission are immune to malaria? It has been proven that individuals develop immunity to the malarial parasites through repeated exposure all year round and over the years. In this type of settings, children over the age of five and adults are progressively more immune to malaria because they have been expos ed to the parasite for a prolonged and constant period of time. Those living in an ar ea of low transmission setting should progress to clinical disease and severe manifestati ons and the age is a less determining factor since no one is constantly exposed to the pa rasite over the course of the his life (Reyburn et al., 2005). In fact, recent studies in Tanzania and Kenya have highlighted the fact that the epidemiology and clinical manifes tations of severe malaria vary with the intensity of transmission (Snow et al., 1994). The oretically, individuals living in an area of low transmission should suffer from complicated disease but this is not always the case. A study conducted in Daraweesh, Sudan, an ar ea of low and unstable transmission where the season of heavy rain is followed by the d ry season, found that local dwellers did acquire the ability to control some infection. However, the protection is far from solid and malaria occurs in all age-groups, althoug h the risk of getting malaria decreases with age (Theander, 1998).

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49 Geographical Distribution Figure 7: Geographical Distribution of Malaria. (W HO 2006) The distribution of malaria depends mainly on clim atic factors such as temperature, humidity, and rainfalls (CDC, 2004). As mentioned early, in tropical and subtropical areas two factors are needed for the tr ansmission of malaria to be successful: I) Anopheles mosquitoes can survive and multiply and II) Malari a parasites can complete their growth cycle in the mosquitoes (extrinsic inc ubation period). There are also some geographical features that pre vent the transmission from occurring: high altitudes cooler seasons in some areas deserts (excluding the oases) some islands in the Pacific Ocean which have no loc al Anopheles species capable of transmitting malaria

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50 The highest transmission is found in Africa south o f the Sahara, where the closeness to the equator makes the climate warmer; in these conditions, transmission is more intense, malaria is transmitted year-round, an d P. falciparum predominates. In cooler regions, transmission will be less intens e and more seasonal. There, P. vivax might be more prevalent because it is more toleran t of lower temperatures. Drug Resistance The World Health Organization defines antimalarial drug resistance as the ability of a parasite strain to survive and/or multiply des pite the administration and absorption of a drug given in doses equal to or higher than those usually recommended but within tolerance of the subject (WHO, 2001). This definit ion was later modified to specify that the drug in question must gain access to the parasi te or the infected red blood cell for the duration of the time necessary for its normal actio n (Bruce-Chwatt et al., 1986). The first scientific reviews in the literature that discussed the emerging problem of drug resistance appeared in the mid ‘80s and reported that Plasmodium falciparum in Central and West Africa was becoming resistant not only to chloroqui ne, but also to all existing alternative treatments except quinine (Ambroise-Thomas & Rossig nol, 1986). Today, drug resistance varies largely depending on the geograph ic region, the drug being used, and the different species of the Plasmodium parasite. In P. falciparum resistance has been observed to almost all currently used antimalarials (amodiaquine, chloroquine, mefloquine, quinine and sulfadoxine–pyrimethamine) except for artemisinin and its derivatives. The geographical distributions and ra tes of diffusion have varied considerably. P. vivax has developed resistance rapidly to sulfadoxine–pyr imethamine in

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51 many areas. Chloroquine resistance is confined lar gely to Indonesia, East Timor, Papua New Guinea and other parts of Oceania though there are also documented reports from Peru. P. vivax remains sensitive to chloroquine in South-East Asia the Indian subcontinent, the Korean peninsula, the Middle East north-east Africa, and most of South and Central America (Table 7 & Fig. 8) (WHO, 2006). Table 7: Distribution of Drug-Resistant P. falciparum Malaria. (WHO, 2001)

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52 Figure 8: Malaria Transmission Areas and Reported D rug Resistance. (WHO, 2004) A number of factors that are thought to contribute to the development and intensification of drug resistance have been identi fied. These are: incorrect dosing, noncompliance with duration of dosing regimen, poor dr ug quality, drug interactions, poor or erratic absorption, and misdiagnosis (WHO, 2001). In terms of the molecular processes that lead to th e formation of resistance, there are a number of mechanisms that characterize each c ategory of drugs. Chloroquine has the capacity of capture harmful hem e moieties which are produced by the parasites as a by-product of hemogl obin digestion, its major source of amino acids. This process occurs in dige stive vacuoles in which choloroquine can exert its function if the parasite is chloroquine sensitive, otherwise, if the parasites are choloroquine resist ant, this drug will not be able to

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53 accumulate at sufficient levels that will allow it to play its role of eliminating heme moieties. The different behavior is due to mu tations in the pfcrt ( Plasmodium falciparum chloroquine resistance transporter) gene; resistan t strains of the parasite have several nucleotide substitutio ns due to the great variety of chloroquine resistant parasites, but one amino acid is found constantly in resistant strains, K76T (Hyde, 2007). These mutations are th ought to play a role in determining the characteristics of CRT (chloroquine resistance transporter) with respect to the drugs; scientists suggest that the K 76T amino acid alters the activity of the CRT in a way that chloroquine exits the vacu ole more rapidly (Johnson et al., 2004). One possible explanation for this modi fication is that when K76 is present, the expulsion of drug outside the vacuole through CRT is limited due to the amino acid positive charge; when the strain is resistant and K76T comes into action, the movement of chloroquine towards the out side of the vacuole is excessive and accelerated (Hyde, 2007). Another category of drugs that is worth mentioning are the antifolates. The principal drugs of interest within this class are p yrimethamine and sulfadoxine. Pyrimethamine targets the dihydrofolate reductase ( DHFR) activity of the DHFRthymidylate synthetase protein, while sulfadoxine i nhibits the dihydropteroate synthetase (DHPS) activity of the DHPS-hydroxymethy lpterin pyrophosphokinase. DHFR enzyme is present in both the host and the parasite and its role pertains the constant supply of fully reduced forms of folate conenzymes that help, for instance, in the provisio n of nucleotides for DNA synthesis and the metabolism of certain amino acids DHPS is only found in the

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54 parasite and it is also used in the biosynthesis of essential folate enzymes. These drugs bind to these two enzymes several hundred tim es more strongly in the parasite than in the human orthologue, therefore ef fectively exerting its action on the parasite and not affecting the functions of the enzyme in humans in the case of DHFR. The genetic basis of resistance to the antif olate drugs is similar to that underlying chloroquine resistance, in that a small number of point mutations in these two target genes appear to be responsible for the major part of resistance (Hyde, 2007). Finally artemisinin is thought to function, upon ac tivation by Fe2+, by inhibiting the Ca2+ sarco/endoplasmic reticulum calcium-dependent ATPa se (SERCA) PfATPase6, a transporter found on membranous struct ures within the parasite cytoplasm (Hyde, 2007). Resistance to artemisinin products has not quite emerged yet, primarily due to its use in combinatio n therapy, even though in vitro resistance has been reported in isolates from Frenc h Guiana, Cambodia, and Senegal. In a study conducted by Jambou and colleg ues, the coding sequences of PfATPase6 isolates from these three countries were established and substantial polymorphism was observed, especially in the Cambod ian strains. More specifically, the polymorphism of S769N, found in m any of the isolates, was associated with an increased mean IC50 (measure of the effectiveness of a compound in inhibiting biological or biochemical fu nction) for arthemeter (Jambou et al., 2005). In fact, for instance, the S769N mutation differs from the engineered L263E replacement, which eliminates arte misinin inhibition of the PfATPase6 enzymatic activity (Uhlemann et al., 2005 ). In certain species,

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55 residue 769 is essential to the structural transiti ons needed for the progress of the ATPase cycle and calcium binding and release. By a nalogy with the inhibition of the rabbit SERCA by thapsigargin, a structural anal ogue of artemisinins, it is inferred that the S769N mutation prevents artemisin in derivatives from interfering with these conformational changes (Jambou et al., 2 005).

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56 Table 8: P. falciparum Proteins with a Proven Role in Resistance to Clini cal Antimalarial Drugs. (Jambou et al., 2007)

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57 Cytochrome b5 Reductase (cb5r) and Cytochrome b5 (cb5) Cytochrome b5 Reductase Cytochrome b5 reductase (cb5r) is a member of the FNR superfamily of flavoprotein oxidoreductases and contains a single flavin adenine dinucleotide (FAD) group (Shirabe et al., 1997). The function of this enzyme is to catalyze the transfer of two electrons from NADH to two molecules of cytochr ome b5 in one catalytic cycle (Strittmatter, 1965). Cb5r exists in two forms, a membrane-bound form that i s comprised of both a hydrophobic membrane-anchoring region (Mr ~ 3 kDa, residues 1-25) and a hydrophilic, catalytic portion (Mr ~ 30 kDa, residues 26-300), and a soluble form compr ising only the soluble reductase. The soluble form of cb5r exists in circulating erythrocytes and participates in methemoglobin reduction, while the membrane-bound form is embedded in the membranes of the somatic cells such as those of the endoplasmic reticulum, mitochondria, and plasma membrane (Bewley et al., 2 001). For the purpose of this project, the membrane-bound form of cb5r will have greater importance and will represent the predomina nt topic. As mentioned, membranebound cb5r consists of a hydrophobic N -terminal membrane-anchoring domain, and a larger hydrophilic, soluble catalytic domain. It c ontains four conserved motifs: two NADH binding motifs, a flavin-binding motif, and a FMN/FAD selectivity motif (Marohnic et al., 2005). A well known cb5r protein structure is represented by the rat cb5r. The structure has a typical two-domain arrangement, with an amino -terminal binding domain (residues 33-147) and a carboxyl-terminal NADH-binding domain (residues 171-300). These two

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58 domains are linked by a 25-amino acid, three-strand ed antiparallel -sheet (residues 148170) that is thought to serve as a hinge to orient the NADH and FAD-binding domains, so that an efficient transfer of electrons from NAD H to flavin can occur. There is also an FAD molecule that interacts with both domains throu gh hydrogen bonds (Fig. 9b). The FAD-binding domain is a six-stranded, antiparallel -barrel with a ~ 30r twist of each strand. The barrel is capped by the only -helix in this domain. A long loop (residues 110-125), located between strand 5 and the -helix, forms a lid that contributes to the majority of interactions of the FAD-binding domain with the adenine dinucleotide moiety of the FAD. The NADH-binding domain contains a typ ical Rossman fold (three // layers that pack into a five-stranded parallel -sheet in the order 3, 2, 1, 4, 5) (Fig. 9a) (Bewley et al., 2001); these folds are able to bind only one nucleotide at a time, thus in this case there are two paired Rossman folds that b ind to the incoming NADH molecule (Rao & Rossman, 1973). Figure 9: Structure of Rat Cb5r. (Bewley et al., 2001) (a) Stereo ribbon diagram of rat cb5r. The FAD-binding domain is colored red, the NADHbinding domain is blue, the three-stranded linker domain is green, and the FAD molecule is shown in a ball and stick

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59 representation using standard atom colors. (b) Inte ractions of the FAD and visible region of the NAD. Water-mediated hydrogen bonds are shown as cyan dot ted lines. Cytochrome b5 Cytochrome b5 (cb5) is an electron transfer protein with a redox potent ial of 20 mV which is capable of accepting and transferring a single electron (Velick & Strittmatter, 1956). It is a small protein of abou t 17 kDa found throughout the phyla, from yeasts to insects, seed plants and animals. I n all species and tissues (except erythrocytes), it is anchored in the membranes by a hydrophobic, carboxyl-terminal end of the peptide chain. In erythrocytes, a truncated soluble form of the protein is found (Schenkman & Jansson, 1999). The protein has a hem e prosthetic group that lacks a free coordination position and consequently, it serves a s a physiological electron transfer component facilitating a number of reactions, inclu ding fatty acid desaturation, fatty acid elongation, and cytochrome P450 (P450) monooxygenat ion (which in turn is involved in the catabolism of xenobiotics) (Schenkman & Jansson 1999; Keyes et al., 1979; Conney et al., 1957). Structurally, cb5 is a small, cylindrical, acidic membrane protein c onsisting of 6 helices and 5 -strands. The protein is folded into two domains, t he larger of which is the cytosolic heme-containing, amino-terminal, hydrophi lic region. The smaller domain is the hydrophobic, membrane-binding carboxyl portion of 14–18 residues, connected to the globular domain by a proline-containing hinge regio n of ~7 amino acids, and followed by 7 polar amino acids at the very end of the polypept ide chain (Fig. 10) (Mathews, 1985). Proteases can clip before or after the proline-cont aining hinge region, releasing the soluble, catalytic heme-containing domain. In mamm als, 23 of the 134 amino acids are

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60 either glutamate or aspartate and are completely co nserved. Most of them are located in the hydrophilic region of the protein. The heme is located in a hydrophobic pocket, and the heme iron is coordinated with two completely co nserved histidine side chains, H68 and H44. This binding prevents direct interaction of cb5 with molecular oxygen (Schenkman & Jansson, 2003). Another important characteristic of cb5 is its site of biosynthesis and subsequent association with the membrane of the organelles in which they are embedded. It has been shown that cb5 and its reductase cb5r are synthesized in free polysomes and therefore, their association with the membrane must be establi shed post-translationally, as opposed to the case of another reductase, P450, which plays a variety of roles in conjunction with cb5 and cb5r. P450 is synthesized in bound polysomes and cotr anslationally inserted into the endoplasmic reticulum membranes (Okada et al., 1981). Figure 10: Cytochrome b5 Diagram. (Ramamoorthy, 2009) This figure shows the hydrophobic portion imbedded in the phospholipid bilayer of the membrane and the larger hydrophilic segment that lays in the cytosol

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61 Cb5r and Cb5 Function Cb5r and cb5 work in an interrelated fashion and depend on each other for the exchange and flow of electrons. More specifically, cb5r catalyzes the transfer of two electrons from NADH to two molecules of cb5 in one catalytic cycle (Shirabe et al., 1997). When these two proteins are found in the so luble form in erythrocytes, the electrons donated to cb5 are used to reduce the ferric heme iron of hemoglo bin to the ferrous state, resulting in the formation of methem oglobin (Hultquist & Passon, 1971). The membrane-bound forms on the other hand, serve a different function, participating in the elongation and desaturation of fatty acids, cho lesterol biosynthesis, and some of cytochrome P450 mediated dug metabolism (Schenkman & Jansson, 1999; Keyes et al., 1979; Conney et al., 1957). Physiological electron donor of cb5 is NADH-cytochrome b5 reductase and acceptors are methemoglobin, fatty ac id desaturase, and cytochrome P450 (Shirabe et al., 1997). In fatty acid desaturation, cb5 participates in the synthesis of unsaturated fatty acids by donating electrons, which it receives from NADH-cb5r, to desaturases which are microsomal, non-heme, iron-containing proteins. Th e desaturases use the electrons from cb5 to generate an electron-deficient, activated oxyge n species which removes electrons from the saturated hydrocarbon. Unsaturated fatty acids possess a structural role in cellular membranes where they greatly contribute to the fluidity of the cellular membranes. They are also precursors of polyunsatur ated fatty acids which are important mediators and regulators of various cell functions (Vergeres & Waskell, 1995).

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62 In cholesterol biosynthesis, cb5 has been shown to provide the reducing equivalents to the 19-step microsomal conversion of lanosterol to cholesterol; more precisely, it supplies electrons in the reaction in which 4-methyl sterol oxidase (a desaturase) oxidizes the C-30 methyl group of lanos terol (the cholesterol precursor) to a steroid acid which can be consequently decarboxylat ed (Fukushima et al., 1981). The role and mechanism of action of cb5r and cb5 in drug metabolism is still subject of much research; many studies link the inv olvement of cb5r and cb5 in cytochrome P450-mediated reactions, which include d rug metabolism (Porter, 2002). The interests are concentrated on the possibility o f an NADH-cb5 driven, P450 reductase independent pathway of drug metabolism (Yamazaky et al., 1996). In particular, the cb5r/cb5 action is thought to be involved in the donation o f the second of the two required electrons for the reduction of P450 in the catalyti c cycle (Pompon & Coon, 1984). The complete process and set of reactions will be expla ined in the next section. Cb5r and Cb5 Relationship and Interaction The link between cb5r and cb5 comes from the knowledge that there exist a transfer of electrons and a consequent electrostati c interaction between the reductase and the hemoprotein (Shirabe et al., 1997). There are propionate groups of the heme in cb5, which are thought to be an electron pathway to heme iron from cb5r, that protrude from the molecular surface. All charged residues around this heme-protruding site are negative (Mathews et al., 1971). These negatively charged residues are very numerous and are able to form several hydrogen bond pairs wi th positively charged residues on the flavin protruding site of cb5r. The actual interaction occurs between some carb oxyl

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63 residues of cb5 have been proven to be involved in the charge-pair ing with the lysil residues of cb5r (Strittmatter et al., 1992). In the same study, it was found that lysil residues around the flavin-protruding site of cb5r are implicated in the formation of charge pairs with carboxylate residues of cb5. The specific links are found between Lys13 and Glu48 (or Glu 56), Lys 97 and Glu43 (or G lu44), and Lys134 and the heme propionate; in all three pairs, the former residue is of cb5r and the latter is of cb5. In figure 11, a docking model of cb5r and cb5, the positively charged residues around the flavin of cb5r are involved in hydrogen bonding with cb5. Here, Lys 97 and Lys134 of cb5r and Glu44 and propionate of cb5 form two hydrogen-bonded pairs. However, Lys13 and Glu48 or Glu56 are separated. A ll three hydrogen-bonded pairs (Lys13/Glu48, Lys 97/Glu43, and Lys134/propionate) cannot be formed simultaneously in a single docking form like this one because the three residues of each of the two molecules are arranged counterclockwise when viewed from the exterior (Nishida & Miki, 1996).

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64 Figure 11: Ribbon Diagram of the Docking Model of P ig Cb5r and Cb5. (Nishida & Miki, 1996) The molecules of cb5r (left) and cb5 (right) are represented by yellow and brown, respe ctively. The residues whose side chains are involved in hydrogen bonds be tween the molecules are represented by stick model and colored by their charge, cyan (positive), red ( negative), and white (neutral). The atoms involved in the hydrogen bonds are represented by balls. Another consideration to be made is that cb5 also interacts with the NADHbinding domain of cb5r through the formation of four salt bridges. Ther e is evidence through a study on transient kinetics, that the binding of NADH modulates the formation of the cb5r/cb5 complex. It is also thought that these two NADH a nd FAD domains are tightly packed under NADH-binding state. Hydrogenbonding of cb5 to both domains of cb5r is reasonable in the view of the modulation of cb5r/cb5 complex formation by the NADH binding (Meyer et al., 1995). In order to prove that the ionic interactions and s pecific chemical bonding between these two proteins are exact, cb5r and cb5 mutants were constructed by means of

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65 bacterial expression and site-directed mutagenesis (Strittmatter et al., 1992). In this study, the amino acids that are believed to interac t are lysine residues K41, K125, K162, and K164 in cb5r and glutamate residues E47, E48, E52, E60 and asp artate residue D64 in cb5. The activity of cb5r mutants was measured with potassium ferricyanide as an electron acceptor; it was found that the activity i n the mutant was almost the same as that of the cb5r wild type. This suggests that electron transfer from NADH to cb5r remains intact in all mutants. Also the specific activity of all cb5r mutants was reported to be considerably less efficient than that of the wild t ype, and the Km values were significantly elevated when the wild type cb5 was used as the electron acceptor (Table 9). Table 9: Kinetic Properties of Cb5r Mutants Using Human Erythrocyte Wild-Type Cb5. (Shirabe et al., 1998) This result may be explained by the fact that the residues from cb5 that are involved in the interaction with cb5r are negatively charged and normally are neutraliz ed by the positive charge of the cb5r residues. The difference in this case is that th e cb5r mutants have lost their positive charge. From thes e results it can be concluded that the four lysine residues K41, K125, K162, and K163 may participate in the electrostatic interaction with cb5 (Shirabe et al., 1998). Based on the same study, cross-linking studies

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66 of the binding of residues E47, E48, E52, E60, and D64 (group A residues) of cb5 with cb5r were undertaken. Cb5 mutants of the glutamate residues were generated. One or all of these residues were changed to Ala. The obtaine d Km and Kcat values were not significantly different than those of the wild type indicating that none of these residues participates in the interaction with cb5r. The role in binding to cb5r by other acidic residues located close to the heme group of cb5 was examined. The substitution of E41, E42, D57, and E63 (group B) with Ala caused a fivefold increase in the Km with no notable change in the Kcat. This indicates that some of the group B residues participate in the interaction of cb5 with cb5r (Shirabe et al., 1998). Role in Drug Metabolism and Involvement in the Deto xification of Xenobiotics through Cytochrome P450 In the past few decades, hundreds of experiments ha ve been performed to study the effect and the mechanism of action of the NADH, NADPH, cb5r, cb5, P450 reductase, and P450 sequence of reactions on the substrate met abolism of P450 (Vergeres & Waskell, 1995). Cytochrome P450 is a mixed functio n oxidase which catalyzes the oxidation of numerous endogenous and exogenous comp ounds (Siegel et al., 2007). The activation of P450 for oxidation of its substrates and activation of molecular oxygen, requires two electrons which are provided by NADPH or, possibly, cb5 (Ortiz de Montellano, 1986). The first electron is transferr ed to P450 by NADPH P450 reductase while the second electron can either be transferred by NADPH P450 reductase or, as speculated, cb5 (Vergeres & Waskell, 1995). Cb5 cannot transfer the first electron to P450 because the potential of cb5 is ~ 20 mV, while that of ferric P450 (that needs t o be reduced to ferrous P450) is ~ -330 mV. Therefore th e reduction of P450 by

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67 NADH/cb5r/cb5 chain of electron flow would be too slow to suppor t the necessities of P450-dependent metabolism of various substances, in cluding drugs (Fig. 12) (Porter, 2002). Figure 12: Electron Transport Pathways to Cytochrom e P450 in the Endoplasmic Reticulum. (Porter, 2002) After being reduced by NADPH and/or cb5, P450 reacts with oxygen to form an active oxygen complex, which in turn hydroxylates the vari ous substrates (Gillette, 1971). The reaction is as follows: NADPH + A(P450) AH2 + NADP AH2 + O2 Active oxygen complex Active oxygen complex + drug Hydroxylated d rug + A + H2O As mentioned, when the source of reducing equivale nts is NADH, the reduction of P450 is very slow, about 10% of the rate with NA DPH, even in the case of the input of the second electron (Shenkman & Jansson, 1999). Ea rly studies on P450 indicated that the rate-limiting step in the monooxygenase reactio n was the input of a second electron. It was also suggested that the role of cb5 was to speed up this electron transfer by providing the second electron more rapidly from NAD H than could be provided from NADPH (Hildebrandt, 1971).

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68 A hypothesis has been presented to explain this sti mulatory effect of cb5. Cb5 forms a two-electron acceptor heterodimeric complex with P450 that causes cb5 to stimulate the substrate oxidation by P450. This wo uld also allow for a single interaction, rather than two, with the reductase to provide the two electrons needed for the activation of molecular oxygen. Cb5 was suggested to act by oxidizing ferrous P450, th ereby allowing the input of a second electron to the oxid ized P450, and then returning the electron to the oxyferrous P450 intermediate. This function of cb5 was suggested to be due to the need for only single interaction with NA DPH-P450 reductase by the heterodimeric complex rather than two separate inte ractions with P450 alone (Shenkman & Jansson, 1999). Phylogenetic Analysis of P. falciparum Cb5r and Cb5 Cb5r and cb5 have a high pharmacological significance due to th eir role in drug metabolism and detoxification of xenobiotics. With the increasing levels of drug resistance to essentially all of our available anti malarial medicines, there is an urgent need to find new parasitic drug targets. A new imp ortant aspect that has not yet been mentioned in this review, is the identification of plant cb5r as a novel herbicidal target (Reindl, 2005), thus making this class of agents as potential candidates for new antiparasitic drugs against P. falciparum malaria. The phylogenetic relationship between plant and P. falciparum cb5r and cb5 is a useful tool to evaluate the evolutionary dist ance. Analyzing the phylogenetic tree can give us preciou s insights into the possible relatedness of these proteins which belong to these two different kingdoms (plantae and protista, respectively).

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69 Plasmodium falciparum cb5r formed a distinct clade with other protozoan, hav ing the closest homology to plants, and all protozoan i soforms were distinct from their mammalian orthologues (Fig. 13). Considering that plant cb5r is an herbicidal target, it can be speculated that P.falciparum cb5r may also represent a potential novel drug target. An additional consideration that can be extrapolate d from the phylogenetic tree is that protozoan cb5r is more closely related to plant cb5r than mammalian cb5r. This is a further advantage in terms of novel drug target and drug efficacy: the closeness in the evolutionary course of protozoan cb5r to plant cb5r may lead to promising treatment outcomes. This is further supported by the fact th at the host cb5r (human) will not be affected by the action of the drug since human cb5r is phylogenetically too far away from its protozoan orthologue. Figure 13: Phylogram of Cytochrome b5 Reductases. Amino acid sequences of cb5r were aligned using ClustalW.

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70 P. falciparum cb5 also formed a distinct clade separate from their m ammalian orthologues (Fig. 13a). The oldest cb5 orthologues are found in prokaryotes, including purple bacteria and Gram-positive species. Figure 13a: Phylogram of Cytochrome b5. Various cb5 amino acid sequences were aligned using ClustalW. Structural Comparison of P. falciparum Cb5r with Mammalian Cb5r and Other Species Several cb5rs from a variety of species were aligned using Clu stalW and Jalview to compare the sequences of the four conserved bind ing motifs (Fig.14a, 14b, 14c). In particular, Plasmodium falciparum and mammalian cb5r were analyzed. In the flavinbinding domain, at the second amino acid position, P. falciparum has a serine (S) while human cb5r has a proline (P); also, in the same domain, at t he sixth position, there is a

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71 valine (V) in P. falciparum cb5r and an isoleucine (I) in the human code. Lastly a tyrosine (Y) in P. falciparum and a serine (S) in human are found at the seventh position. There are a total of three substitutions in amino a cids in the flavin-binding domain sequence (which consists of seven amino acids) betw een P. falciparum and human cb5r. P. falciparum cb5r only shares two differences with the other mammal ian species presented here: Pro becomes Ser and Ser becomes Tyr in mammalian and P. falciparum respectively. Compared to many plant and parasite species, P. falciparum cb5r shares many differences, especially in the first two and l ast two amino acids. P. falciparum cb5r is the also the only protein with a Tyrosine at the seventh position in the flavin-binding domain. The third, fourth, and fifth amino acid in the domain are conserved throughout the species (Fig. 14a). Figure 14a: Amino Acid Sequence Alignment with the First Two Binding Motifs by ClustalW Analysis and Jalview. Comparison of mammalian with P. falciparum cb5r shows three amino acid substitutions in the FADbinding motif: Pro Ser, Ile Val, and Ser Tyr. The 9-amino acid gap preceding the FAD/ FMN selectivity motif that is present in many fungal an d plant cb5rs, is reduced to a 2-amino acid gap in P.

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72 falciparum This gap is absent in mammalian cb5r. This gap may decrease the stability of the prot ein and could serve as a new target for the development of anti-parasitic drugs. The first NADH-binding domain has the signature seq uence GxGxxP. The sequences in mammalians are very conserved, all bei ng GTGITP, while P. falciparum has GTGMTP, with only one substitution from Isoleuc ine (I) in mammalians to Methionine (M) in the parasite. The presence of Me t at the fourth position is unique to P.falciparum (Fig. 14b). Figure 14b: Continued Amino Acid Sequence Alignment Containing the First NADH-binding Motif by ClustalW Analysis and Jalview The NADH-binding motif is very conserved and has th e signature sequence GxGxxP. In all mammalians the sequence is GTGITP, while the P. falciparum one is GTGMTP. Here the only substitution is Ile t hat becomes Met in P. falciparum The presence of Met at the fourth position is uni que to P.falciparum There is a 16-amino acid gap preceding the NADH-binding m otif in fungi and plants that is reduced to a 5-ami no acid gap in P. falciparum This gap is also seen as a potential novel drug t arget. There is a 9-amino acid gap preceding the FAD/FMN s electivity motif in many fungal and plant species which corresponds to a “li d” that covers the FAD prosthetic

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73 group in all mammalian and Trypanosoma species ( T. brucei and T. cruzi ), and in Leishmania infantum as well. This variation in the protein structure may decrease the stability of the molecule and could be consider as a target in the design of drugs. This gap in P. falciparum cb5r is made up of only two amino acids so its propert ies should be investigated (Fig. 14a & 15). Figure 14c: Continued Amino Acid Sequence Alignment with the Second NADHbinding Motif by ClustalW Analysis and Jalview. This second NADH-binding domain is extremely conser ved throughout the organisms. Mammalian and P. falciparum cb5r possess exactly the same sequence in the NADH-bin ding domain. All possible sequences are also presented in bold below the Jalview alignm ent. There is another larger 16-amino acid gap in the s equence preceding the NADHbinding motif in all fungal and plant species. Thi s gap is absent in all mammalian

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74 species. This region connects the NADHand FAD-bi nding motifs and it is represented by two -sheets which are missing in those species that lac k this 16-amino acid sequence. This also decreases the stability and catalytic eff iciency of the protein and may constitute a potential target in the development of novel drug s. There is only a 5-amino acid sequence gap in the structure of P. falciparum cb5r (Fig. 14b & 15). Figure 15: The Ribbon Model of Rat and Candida Cb5r This model compares a mammalian (rat) cb5r to a fungal (Candida) cb5r. In the absence of a ribbon model of P. falciparum cb5r, it shows the lack of the “lid” (9-amino-acid gap ) over the FAD prosthetic group in Candida (only partial in P. falciparum ) and the lack of two -sheets (16-amino acid gap) in the region that connects the NADHand FAD-binding motifs in Candid a (only partial in P. falciparum ). The amino acid sequences of the species that were a ligned and analyzed by ClustalW and Jalview were also evaluated by separat ing them into their catalytic domains. From this separation, phylograms were cre ated that reflected the evolution of the individual separated domains. The first amino acid sequence contains the first two domains (NADH-binding domain and the FAD/FMN select ivity domain) and the second amino acid sequence includes the third and fourth d omains i.e. the first and second NADH-binding domain.

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75 Cytochrome b 5 Reductases Amino Acid Sequence Containing the FADbinding Domain and the FAD/FMN Selectivity Domain (Underlined are the Two Domains) >Human -CytB5Red AAP88823.1 [ Homo sapiens ] MGAQLSTLGHMVLFPVWFLYSLLMKLFQRSTPAITLESPDIKYPLRLIDRE IISHD TRRFRFALPSPQHILGLPVGQHIYLSARIDGNLVVRPYTPIS SDDDKGFVDLVIKV YFKDTHPKFPAGGKMSQ YLESMQIGDTIEFRGPSGLLVYQGKGKFAIRPDKKSNP IIR >Chimp -CytB5Red XP_001171082.1 [ Pan troglodytes ] MGAQLSTLGHVVLFPVWFLYSLLMKLFQRSTPAITLESPDIKYPLRLIDRE IISHDT RRFRFALPSPQHILGLPVGQHIYLSARIDGNLVVRPYTPVS SDDDKGFVDLVIKVY FKDTHPKFPAGGKMSQ YLESMQIGDTIEFRGPNGLLVYQGKGKFAIRPDKKSNP VIR >Rat -CytB5Red P20070 [ Rattus norvegicus ] MGAQLSTLSRVVLSPVWFVYSLFMKLFQRSSPAITLENPDIKYPLRLIDKE IISHDT RRFRFALPSPQHILGLPIGQHIYLSTRIDGNLVIRPYTPVS SDDDKGFVDLVVKVYF KDTHPKFPAGGKMSQ YLENMNIGDTIEFRGPNGLLVYQGKGKFAIRADKKSNPV VR >Dog -CytB5Red ABA12483.1 [ Canis familiaris ] MGAQLSTLGHVVLSPVWFLYNLLMKLFQRSTPAITLESPDIKYPLRLIDKE VINHD TRRFRFALPSPQHILGLPVGQHIYLSARIDGNLVIRPYTPVS SDDDKGFVDLVIKV YFKDTHPKFPAGGKMSQ YLESMKIGDTIEFRGPNGLLVYQGKGKFAIRPDKKSN PIIK >Chicken -CytB5Red XP_416445.1 [ Gallus gallus ] MGAQLSTLGWVVTYPLWLIYSTIRRLFGSPRPAITLKDPEVKYALRLIDKE EVSH DTRRFRFALPSVDHVLGLPIGQHIYLSARIDGALVVRPYTPIS SDDDKGFVDLVIK VYMKGVHPKFPDGGKMSQ YLDNLKIGDTIDFRGPSGLLVYKGKGEFAIRPEKKA DPVTK >Xenopus -CytB5Red AAH45265.1 [ Xenopus laevis ] MGAQLSTVSRILTSPFWFIFSIFQRFFGKPRPAITLESPDIKYALRLIDRE EISHDTRR FRFALPSPEHVLGLPIGQHIYLSARVDGNLVVRPYTPVS SDDNKGYVDLVVKIYF KNVHPKFPEGGKMSQ YLDSLRKDETIDFRGPSGLLVYSGKGTFQIRPDKKSPPVP K

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76 >Zebrafish -CytB5Red AAQ97765.1 [ Danio rerio ] MSYAMSTTVAVTVGVVLVSTAGLLGYYYFNRKRKILITLIDPSEKYKLRLV DKEI ISHDTRRFRFALPSPEHVLGLPVGKHVYLSARIDGNLIVRPYTPVS SDDDKGFVDL VVKIYFRDVHPKFPEGGKMSQ YLESLRIGDVIDFRGPGGLLEYKGAGRLDIQADK KAPAETK >Drosophila -CytB5Red AAF50004.1 [ Drosophila melanogaster ] MTEFDFVPLAVGVVAVLAGALIVHYLLNKKSTKPRREPNRTARLRTLVDPN DKY LLPLIEKENLSHDTRRFRFGLPSKQHVLGLPVGQHIHLIATIDNELIIRPY TPIS SDE DVGYVDLVVKVYFKDSHPKFPAGGKMTQ HLEQLELGDKISFRGPSGRLQYLGN GTFSIKKLRKDPPKHV >Caenorhabditis_elegans -CytB5Red AAB66010.1 [ Caenorhabditis elegans ] MVENNTLAITGGVVLISSVSLFLYLRQLRAEKKSKRTLEDDSVKYLLPLIE KFEIS HNTRKFRFGLPSKDHILGLPIGHHVYLSANIGGKLIVRSYTPVS CDLDLGYVDLM VKVYFKNTHERFPDGGKMSQ HLESLKIGDTVSFRGPHGSIIYKGSGLFTVRMDK KAEPKNR >Saccharomyces_cerevisiae_CBR1_[ER] ( Z28365 ) 322aa ScCBR1 MYKYSYYIRRKNEREKKVLKVCIQLALQQETQSIKQSKMAIDAQKLVVVIV IVV VPLLFKFIIGPKTKPVLDPKRNDFQSFPLVEKTILTHNTSMYKFGLPHADD VLGLPI GQHIVIKANINGKDITRSYTPTS LDGDTKGNFELLVKSYPTGNVSK MIGELKIGDSI QIKGPRGNYHYERN >Saccharomyces_cerevisiae_MCR1_[Mito] -( Z26877 ) 302aa ScMCR1 MFSRLSRSHSKALPIALGTVAIAAATAFYFANRNQHSFVFNESNKVFKGDD KWID LPISKIEEESHDTRRFTFKLPTEDSEMGLVLASALFAKFVTPKGSNVVRPY TPVS D LSQKGHFQLVVKHYEGGKMTS HLFGLKPNDTVSFKGPIMKWKWQPN >Candida_albicans_CBR1_[ER] –CaCBR1 chr4 (294aa) EAK92238 [ Candida albicans SC5314 ] MSETTTVPPIETVSEPNPFIVFATVATIISAFIGYYFLQQSKKHTPVLKPD EFQKFPL IEKIRVSHNSAIYRFGLPKSTDRLGLPIGQHISIGATIDGKEVVRSYTPIS TDDQLGH FDLLIKTYENGNISR HVAGKNVGEHIEIRGPKGFFTYTPN >Candida_albicans_MCR1_[Mito] –CaMCR1 chr6(301aa) XM_705739 [ Candida albicans SC5314 ] MLTHHLSKLATPKFLVPFAGATALSIGLALQYSTSNNYIANETGKTFTDSN EWVD LKLSKSIDLTHNTKHLVFKLKDENDVSGLITASCLLTKFVTPKGNNVIRPY TPVS D VNQSGEIDFVIKKYDGGKMSS HIFDLKEGETLSFKGPIVKWKWEPN >Candida_glabrata –CytB5Red XM_448811 [ Candida glabrata CBS138 ] MDGIKILATFSVLVLFYKLFTYSKKGGVSQKEAVKALLKTEFREFELVEKE QLTH NTAKYKFKLADESHVLGLPIGQHITVKTIIGGKPVSRSYTPTS LDEECVGFFELLV KSYPEGNISK HIGDMKIGEKINISGPRGFYEYVPN

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77 >Cryptococcus_neoformans_CBR1_[ER] –CnCBR1(AAW46852 294 aa, C. neoformans JEC 21 strain, chr 13) MFTIEVLAQKLAPHASFLGGLVVAAILGLFIFFQEKDRKVLDPVEWRSFKL VDKD HLSHNTALYRFALPRASDSLGLPIGQHISVAAEINGKQVVRSYTPTT LDDDKGHF DLVVKTYEKGNISR YLSLLTIGQEIKVKGPKGKFVYTPN >Cryptococcus_neoformans_MCR1_[Mito] –CnMRC1(XM_57 2314 352 aa, C. neoformans JEC 21 strain, chr 8) MAAARFLSSARIARPSVISRTAAQYRGYSSAAPSGGANWPLLLSVAGATGV GAY AYLQYNPSVKKEVEAKIKGAEAEKAAAERTAAGISAFVKDTWIPFTLEKVG KYN HNTNIYHFSFGEEGKDKISGGEVASVVLLRSPEGPEQIKDEKDKPIIRPYT PIS PPD QKGSIEFMIKSYSGGKFTP FLSNLSPGQQVLFKGPLQKFKYQPN >Mortierella -CytB5Red ( AB020034 ) [Mortierella alpine] MTLSNPAIAAASGVILAGAYLIDPSALPFVAAGVAATWARVLFKKTAVKTP PMD PKEYRKFKLVDKVHCSPNTAMYKFALPHEDDLLNLPIGQHISIMANINGKD ISRS YTPTS SSDDVGHFVLCIKSYPQGNISK MFSELSIGDSINARGPKGQFSYTPN >Physarum_[slime_mold] -CytB5Red ( AB259870 ) [Physarum polycephalum] MTVTQLLTSLSFEAKLGIVIAAAAVVVISKFAFGSSSSKREPALNPNEYKK FMLRE KQIINHNTRLFRFNLHHPEDVVGLPIGQHMSVKATVDGKEIYRPYTPVS SDDEKG YFDLIIKVYEKGQMSQ YIDHLNPGDFLQVRGPKGQFDYKPN >Leishmania_infantum -CytB5Red ( L.infantum clone JPCM5 MCAN/ES/98/LLM877; 308 aa) MVGVLVIIAFSMAAFFAFMFTRTTKVAMDPTMFKHFKLIKRTEVTHDTFIF RFAL ENETQTLGLPIGQHIVLRADCTTAGKTETVTHSYTPIS SDDEKGYVDFMIKVYFA GVHPSFPHGGRLSQ YMYHMKLGEKIEMRGPQGKFIYLGNGTSRIHKPGKGIVTE >Leishmania_braziliensis -CytB5Red ( XM_001563404 ) [MHOM/BR/75/M2904; 275aa] MQQSEHLAASCAKAPVNTFTSDEYKPFKLISSRYESHDTRRFYFALDSADD SFYM PVASCIIAKYTDADGKDVARPYTPIS SNSTKGHFELLVKKYPKGKMGN HLFAMQ PGDELLIKGPFEKFAYKPN >Leishmania_major -CytB5Red ( XM_001685505 ) [strain Friedlin; 338aa] MAKFMTFGAATALGAAFHTYTSSQRMAAECAAKKEAFTTKFKPFVLGEVLN LA EDVAIFRFLLNDPSDVFDLVPCSTLQAHFKEGANMVDQPMRFYTPIT PNGTKGYF DLLVKKQRPGRFTE HLFSMDVGESLLFRAIQYKLTYKKN >Trypanosoma_brucei -CytB5Red ( XP828456 ; 307 aa, TREU 927/4 GUTat 10.1 clone) MMLAVIVAFAVIFFVTLALAGRGLLPFYRYPPIALNPDVYQSFKLVKKTRV THDS FIFRFALHASHQCLGLPTGHHIRFRVASKHNFTGTPQVVQHSYTPIS SNDDKGFVD FLVKIYYKGSNPAFPNGGRLSQ HLDSLSIGEAVEMLGPVGKFQYMGNGDYTVE MGKGEVKRQ

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78 >Trypanosoma_cruzi -CytB5Red ( XP807822 ; 306 aa, CL Brener strain) MLTYVVAAVIIVLTVFLFFKKGVFFSSSMGVALDASKFQNFKLVDKITVSH NSFIF RFALHSPTQRLGLPIGQHLHIRCMTTNPDGKPEMVQHAYTPVS SDDDLGHVDFLI KVYFKNVHPNFPNGGRLSQ HLYDLPLGTMVEIRGPVGNFEYLGKGNYTVKDGK GKLKKM >Plasmodium_falciparum –CytB5Red ( XP_001350386; 362 aa, strain 3D7) MKRSFRSMLRSIFMRILYFLTSNISSIIIFCVSISFLGYFGKELHNNNKLF NLYSKKH EYEELDTKEKSQEKPFLNGKNQSFKLYKIIKLTPTVKIFIFSYPDEYEYLG LGICKH IKFNASNIEGKIKGKWNNNDDKEKNLKQISRSYTPVY IDKKKKHVHFIIRVYYPD DEYIDGGKMSI QLNKLNNNDEIDINGPFGLLEYKGNNELLHLSKSVK >Arabidopsis -CytB5Red (Arabidopsis thaliana, NM_121783 ) MDTEFLRTLDRQILLGVFVAFVAVGAGAAYFLTSSKKRRVCLDPENFKEFK LVK RHQLSHNVAKFVFELPTSTSVLGLPIGQHISCRGKDGQGEDVIKPYTPTT LDSDVG RFELVIKMYPQGRMSH HFREMRVGDHLAVKGPKGRFKYQPG >Squash -CytB5Red (Cucurbita maxima, AF274589 ) MAAFLRRLATAAPALRYNALCGQSRIESSKFRFPIGTITAVTAGFSYMFYA STSNL VHLAPNCEEDGQKVALKPDKWIEFELQDVARVSHNTNLYRFSFDPSENWGW ML LHASLQELQLIKTNRGEVKYVARSYTPIS DPEAKGYFDLLIKIYPQGKMTQ HFAK LKPGDKLEVKGPIRKLKYSPN >Maize -CytB5Red (Zea mays, AF077372 ) MDFLQEQRFYSVETTVAVAVAVAAVAAGGAFLLLRSRKPKGCLDPENFRKF KL VEKKQISHNVARFKFALPTPTSVLGLPIGQHISCRGQDATGEEVIKPYTPT TLDSD LGYFELVIKMYPQGRMSHHFREMKVGDYLSVKGPKGRFKYH >Rice -CytB5Red (Oryza sativa (japonica cultivar-gr oup), NM_001051147 ) MDLLHGESVQTTVAIAVAVVAVAAGGAFLLLRSRKPKGCLDPENFKKFKLV EK KQISHNVARFKFALPTPTSVLGLPIGQHISCRGQDATGEEVIKPYTPTTLD SDLGH FELVIKMYPQGRMSHHFREMKVGDYMSVKGPKGRFRYQ >Chlamydomonas_[green_alga] -CytB5Red (Chlamydomona s reinhardtii(green algae), XM_001695672 ) MRSVITLLSYWQVGAGLLVVLVLIQALVFLRKKTKKPFLDPSEFQPVPLVE KTLIT HNTVRLRFALPDPEQRVGLPIGQHISFKAQGEDGKDVIRPYTPVSDDDQLG AVDF VIKLYPTGKMSQVIAKMQLGDTMLMKGPKGRFTYT >Tung-oil_Tree -CytB5Red (Vernicia fordii, AY819697 ) MDLEFLQTLDVQILVGVAVAVLAIGIGAVFLFSSKKPKGCLDPENFKDFKL VNRT QLSHNVAKFSFALPTPTSVLGLPIGQHISCRGKDSQGEEVIKPYTPTTLDS DVGHF ELVIKMYPQGRMSHHFREMRVGDYLSVKGPKGRFRYQ

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79 The phylogram below (Fig. 16) depicts the evolution ary distance among all the different species based on the first catalytic doma in containing the FAD-binding domain and FAD/FMN selectivity domain. P. falciparum and other parasites belong to a clade different from the mammalian species and have the c losest homology to fungal species. Also plants form a distinct clade which is very far from the mammalian one, reflecting a high evolutionary distance and a high degree of ami no acid substitution between the two groups. Figure 16: Cb5r Phylogram According to the First Half of the Prot ein Containing the FAD-binding Domain and the FAD/FMN Selectivity Domain. P.falciparum along with other parasites and mammalian belong to two distinct clades. The closest proximity for P. falciparum is to fungal species. A high evolutionary distance between plants and mammalians is also observed in this phylogram.

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80 Cytochrome b 5 Reductases Amino Acid Sequence Containing the Firs t and Second NADH-binding domain (Underlined are the Two Domains ) >Human -CytB5Red AAP88823.1 [ Homo sapiens ] TVKSVGMIAGGTGITP MLQVIRAIMKDPDDHTVCHLLFANQTEKDILLRPELEEL RNKHSARFKLWYTLDRAPEAWDYGQGFVNEEMIRDHLPPPEEEPLVLMCGP PP M IQYACLPNLDHVGHPTERCFVF* >Chimp -CytB5Red XP_001171082.1 [ Pan troglodytes ] TVKSVGMIAGGTGITP MLQVIRAIMKDPDDHTVCHLLFANQTEKDILLRPELEEL RNEHSARFKLWYTLDRAPEAWDYSQGFVNEEMIRDHLPPPEEEPLVLMCGP PPM IQYACLPNLDRVGHPKERCFAF* >Rat -CytB5Red P20070 [ Rattus norvegicus ] TVKSVGMIAGGTGITP MLQVIRAVLKDPNDHTVCYLLFANQSEKDILLRPELEEL RNEHSSRFKLWYTVDKAPDAWDYSQGFVNEEMIRDHLPPPGEETLILMCGP PPM IQFACLPNLERVGHPKERCFTF* >Dog -CytB5Red ABA12483.1 [ Canis familiaris ] TVKSVGMIAGGTGITP MLQVIRAIIKDPHDPTVCHLLFANQTEKDILLRPELEELR NEHSARFKLWYTVDKAPEAWDYSQGFVNEEMIRDHLPPPEEEPLILMCGPP PM I QYACLPNLDRVGHPKERCFAF* >Chicken -CytB5Red XP_416445.1 [ Gallus gallus ] KVKYVGMIAGGTGITP MLQIIRAIMKDKDDGTVCQLLFANQTEKDILQRSELEEI QVQHPNRFKCWYTLDKAPENWDYSQGFVNQDMIRDHLPPPQSDVLILMCGP PP M IQYACIPNLDKLGYAKDMRFAF* >Xenopus -CytB5Red AAH45265.1 [ Xenopus laevis ] KANHLGMIAGGTGITP MLQLIRAILKDKEDKTICYLLFANQTEKDILLRSELEEIR ANHPSRFKLWYTLDRAPEDWDYSQGFVNEDMISSFMPPPGDDVLILMCGPP PM V QYAINPSLDKLSYPQDRRFAY* >Zebrafish -CytB5Red AAQ97765.1 [ Danio rerio ] TVKSLGLIAGGTGITP MLQLIRDITKNPNDTTTCSLLFANQTEKDILLKDELEEIQA RHSDRFKLWFTVDRAPADWEYSQGFISAEMIQDHLPPPSDDSMILMCGPPP M IQF ACNPNLDKLGYRQSQRFAY* >Drosophila -CytB5Red AAF50004.1 [ Drosophila melanogaster ] TAKRVNMIAGGTGITP MLQLAREVLKRSDKDKTELALLFANQSEKDILLRAELDE LAQKHPDQFKIWYTVDKANEGWQYSVGFINEEMIAAHLLPAKDDTIVLLCG PPP M INFACNPALDKLGYHPDTRFAY* >Caenorhabditis_elegans -CytB5Red AAB66010.1 [ Caenorhabditis elegans ]

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81 FFKHLSMIAGGTGITP MLQVIAAILRDPIDATQIRLLFANQTEDDILCRKELDELAE KHPTRFRVWYTVSKASKDWRYSTGHINEEMIKEHLFPSNEESAVLLCGPPA M IN CACIPNLDKLGHNSENYLIF* >Saccharomyces_cerevisiae_CBR1_[ER] ( Z28365 ) 322aa ScCBR1 CRSHLGMIAGGTGIAP MYQIMKAIAMDPHDTTKVSLVFGNVHEEDILLKKELEA LVAMKPSQFKIVYYLDSPDREDWTGGVGYITKDVIKEHLPAATMDNVQILI CGPP AM VASVRRSTVDLGFRRSKPLSKMEDQVFVF* >Saccharomyces_cerevisiae_MCR1_[Mito] -( Z26877 ) 302aa ScMCR1 QFKSITLLGAGTGINP LYQLAHHIVENPNDKTKVNLLYGNKTPQDILLRKELDAL KEKYPDKFNVTYFVDDKQDDQDFDGEISFISKDFIQEHVPGPKESTHLFVC GPPPF MNAYSGEKKSPKDQGELIGILNNLGYSKDQVFKF* >Candida_albicans_CBR1_[ER] –CaCBR1 chr4 (294aa) EAK92238 [ Candida albicans SC5314 ] MVKSFGMIAGGTGIAP MYQIITAILKNPEDKTKIHLVYANVTESDILLKEELDNFA ARHPDRLKIHYVLNEAPANWQGSVGFVTPEIIDTHLPKASNDTNLLLCGPP PM VS AMKKAAVELGFQKAKPVSKLGDQVFVF* >Candida_albicans_MCR1_[Mito] –CaMCR1 chr6(301aa) XM_705739 [ Candida albicans SC5314 ] QFKSIALIGGGTGITP LYQLLHQITSNPKDNTKVNLIYGNLTPEDILLKKEIDAIASK HKDQVKVHYFVDKADEKKWEGQIGFITKEFLQKELEKPGSDFKVFVCGPPG L YK AISGPKVSPTDQGELTGALKDLGFEKEHVFKF* >Candida_glabrata –CytB5Red XM_448811 [ Candida glabrata CBS138 ] VHKHLAMVAGGTGITP MFQIMKAIARDPSDKTRVTLLYGNVLEEDILLKQELDD LVKQRPDQFKITYLLDKPERDDWEGGVGYVTLDLMKESFPSAEEDVQLLVC GPP GM VSSVKRNAVALGFPRAKPVSKMEDRVFVF* >Cryptococcus_neoformans_CBR1_[ER] –CnCBR1(AAW46852 294 aa, C. neoformans JEC 21 strain, chr 13) MAPHLVMIAGGTGITP MYQIIKSSIKTPGDKTRLSLIYANIQEDDILLKKEIDELQA KSNGRFDVKYVLNNPPEGWTGGVGFVTKEMIEEAMPSSGVGSANHGEGHKV L MCGPPPM ITAMKGHLAQIGYPAPRSVSKLEDQVFLF* >Cryptococcus_neoformans_MCR1_[Mito] –CnMRC1(XM_57 2314 352 aa, C. neoformans JEC 21 strain, chr 8) SFEKGLCIAGGSGITP MWQLINHSLSIPEDKTKWTLIYSNVSEADILLRKEFDALA QKYPGRLDIKYVLDKGPWGWKGETGYVTADLIKKTFPKNEGENIRAFVCGP PGQ MKAVSGEKDGMKQGELAGALKELGYTSDEVFKY* >Mortierella -CytB5Red ( AB020034 ) [Mortierella alpine]

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82 MCRAIGMIAGGTGLTP MLQIIRAIVKNPEDKTQVNFIFANVTEEDIILKAELDLLSQ KHPQFKVYYVLNNAPEGWTGGVGFVNADMIKEHMPAPAADIKVLLCGPPPM VS AMSKITQDLGYDKVNAVSKLPDQVFKF* >Physarum_[slime_mold] -CytB5Red ( AB259870 ) [Physarum polycephalum] MVKEMGMIAGGTGITP MLQVARAIIKNPKEKTIINLIFANVNEDDILLRTELDDM AKKYSNFKVYYVLNNPPAGWTGGVGFVSADMIKQHFSPPSSDIKVMMCGPP MM NKAMQGHLETLGYTPEQWFIF* >Leishmania_infantum -CytB5Red ( L.infantum clone JPCM5 MCAN/ES/98/LLM877; 308 aa) KVDAYAAIAGGTGITP ILQIIHAIKKNKEDPTKVFLVYGNQTERDILLRKELDEAA ANDSRFHVWYTIDREATPEWKYDIGYVCEEMFRKHLPVPDMLGSDSVPQNA GI KKVMALMCGPPPM VQMAIKPNLERIGYTADN >Leishmania_braziliensis -CytB5Red ( XM_001563404 ) [MHOM/BR/75/M2904; 275aa] MWKHVGMIAGGTGIAP MYQVIRAVLENPKDKTNISLIYASNQRRDILLANELIEM QKIYNNFNMYLTLLEVPHRWLGGIGYVNSAMVTTFMPKPGEKNTKILVCGP PPM MQAISGDKLFEPGKPPQQGPVGGLLETLGYKEDQVFKY* >Leishmania_major -CytB5Red ( XM_001685505 ) [strain Friedlin; 338aa] RWTDVGMICGGTGLCP ILQFMNASLETEGDSTRLNMLFANRSEKKILLKGLLDE KAREHKDRLQIFYTVDNFDDPDIIANPVYKKGAPLTVDVASQPSTSPDGRK YFHG FKGYIDVPMLQATMPRPSPSTLLLVCGPDPM MVKVVGAAPMVLRAMSGGLAY QPSGAVLNNAADVSGFLGTLGYQKEMVYRF* >Trypanosoma_brucei -CytB5Red ( XP828456 ; 307 aa, TREU 927/4 GUTat 10.1 clone) HVAGFAMVAGGTGITP MMQIIHAILKSPEDPTRLWLVYSNHTEEDILLRDALAEA CKDPRVKVWHTLTRSAPPDWAYGRGRVNEEMLRTHLPPPQLEEGSVTVLLC GP PLM LQDAVKPNLLNIGYSQDNIFTF* >Trypanosoma_cruzi -CytB5Red ( XP807822 ; 306 aa, CL Brener strain) HTDAFTMVAGGTGITP MMQLIRAIMKNKEDRTNIFLVYANQTEDDILLRKELDD CAKDPRMKLWYMLDRETSPQWKYGTGYVDEATLRAHVPVLQPRNSDYNRVVALMCGPPPM LQKAVKPNLEKLGYTAEDMFSF* >Plasmodium_falciparum –CytB5Red ( XP_001350386; 362 aa, strain 3D7) IKKHIVMIAGGTGMTP FFRLINHLLLTKEKELPSDPVYITFIYANRNENEILLKSIFD DYENRFENFKRVYSVDKCLNTNQMGNFENIGFINEELLRKYVLKYEKLNIE VKN KDTLILLCGPPPM TSSIKSILKDQIHMENIIVF* >Arabidopsis -CytB5Red (Arabidopsis thaliana, NM_121783 ) QFRAFGMLAGGSGITP MFQVARAILENPTDKTKVHLIYANVTYDDILLKEELEGL TTNYPEQFKIFYVLNQPPEVWDGGVGFVSKEMIQTHCPAPASDIQILRCGP PPM N KAMAANLEALGYSPEMQFQF*

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83 >Squash -CytB5Red (Cucurbita maxima, AF274589 ) MKKHIGMIAGGTGITP MLQVIDAIAKNQDDITQVSLIFANVSADDILLKEKLDKL AACHPNIKVFYTVSNPPRGWKGGKGHVSKDMIIKCLPSPGNDALILVCGPP GM M KHICGPKNKDFTQGELGGLLKDLGYSKDMVFKF* >Maize -CytB5Red (Zea mays, AF077372 ) VGQVRAFGMLAGGSGITP MFQVARAILENPNDNTKVHLIYANVTYEDILLKDEL DDMAKTYPGRFKIYYVLNQPPENWNGGVGFVSKEMIQSHCPAPAEDIQILR CGP PPM NKAMAAHLDELNYTKEMQFQF* >Rice -CytB5Red (Oryza sativa (japonica cultivar-gr oup), NM_001051147 ) VGQVRAFGMLAGGSGITP MFQVARAILENPNDITKVHLVYANVTHDDILLKEEL DNMAKTYPDRFKIYYVLNQPPEVWNGGVGFVSQDMIKAHLPAPAEDIQILR CGP PPM NKAMAAHLDELGYTKEMQFQF* >Chlamydomonas_[green_alga] -CytB5Red (Chlamydomona s reinhardtii(green algae), XM_001695672 ) PNMVKHFGMLAGGTGITP MFQVLNAILKNPRDTTSVTLLYGNLTEEDILLRKELD ELVAMHGNRLTVYHVLNTPPVDKEWSGGSGFISSELIRTKFPAPSSDIMTL RCGPS PM MVAMEKALTDLGYAEDKQFQF* >Tung-oil_Tree -CytB5Red (Vernicia fordii, AY819697 ) PGQVRAFGMLAGGSGITP MFQVARAILENPNDKTKVYLIYANVTYEDILLKQEL DGLAANYPDRFKVYYVLNQPPEVWDGGVGFVSKEMIENHCPAPASDIQILR CGP PPM NKAMAAHLEALDYTSDMQFQF* The second half of the amino acid sequences of var ious cb5r containing the two NADH-binding domains presents interesting differenc es in terms of evolutionary distance compared to the cb5r first half (Fig.17). P.falciparum has the greatest evolutionary distance from human. Its closest homo logy is to other protozoan and fungal species. Compared to the first half of the protein this phylogram shows less ramifications and more highly grouped clades, sugge sting that there has been a higher degree of conservation in the evolution of this two binding domain-containing part of the protein.

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84 Figure 17: Cb5r Phylogram According to the Second Half of the Pro tein Containing the First and Second NADH-binding Domain. This phylogram assumes a very large evolutionary di stance between this section of cb5r in P. falciparum and human. The closest homology for P. falciparum i s to protozoan and fungal species. There is also a higher degree of evolutionary conservation in this segment of the protein.

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85 OBJECTIVES Cytochrome b5 reductase and its electron acceptor cytochrome b5 have been studied extensively over the last fifty years and a great variety of aspects has been investigated, leading to a plethora of information in the literature. Their known roles in fatty acid desaturation and elongation, cholesterol biosynthesis, and cytochrome P450mediated detoxification of xenobiotic in many diffe rent organisms, ranging from mammalians to fungi and plants, have been explored by many scientists. In a time in which resistance to available medicine s is emerging, and affecting the cure of numerous infectious agents, there is a need to discover new and promising therapeutic targets. In particular malaria treatme nt is experiencing an unprecedented crisis in terms of efficacy, safety, and availabili ty of antimalarial drugs. Resistance to the most feared and aggressive form of malaria that is caused by Plasmodium falciparum has developed to most of the available drugs, with the exception of Artemisinin. The structure, function, biochemical and pharmacologica l properties (in particular the role in the detoxification of xenobiotics), and the interac tion with other cytoplasmic proteins of P. falciparum such as cytochrome b5 reductase and its electron acceptor cytochrome b5 should be investigated. Cb5r and its electron acceptor cb5 may serve as potential drug targets but yet, there are no formal studies that have addressed thi s possibility. An elucidation of the mechanisms and processes in which it is involved ma y generate important information about the possible role of these two proteins as dr ug targets. Interestingly, phylogenetic analysis has revealed that P. falciparum cb5r is closely related to plant cb5r, and plant cb5r

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86 has been identified as a novel herbicidal target. If we are to base our decision on weather or not cb5r would be a fortunate target by comparing its posi tion in the phylogenetic tree to that of plant cb5r, we would likely be prompt to give an affirmative answer. In this project, an analysis of the amino acid sequ ence, binding domains, and protein structure was undertaken. Furthermore, clo ning of P. falciparum cytochrome b5 reductase and cytochrome b5, and the optimization of recombinant protein expre ssion in E. coli was performed. A further objective of this study is the biochemical and kinetic characterization of P. falciparum cytochrome b5 reductase in order to elucidate its enzymatic properties. Here, a comparison with mamm alian cb5r is necessary in order to identify differences in biochemical and kinetic act ivity. A conclusive and useful aim of this study (that has not been investigated here) wo uld be the pharmacological characterization of P. falciparum cb5r through the use of potential inhibitors of this enzyme. There are a number of inhibitors that have been found to alter the enzymatic activity of human cb5r, but none of them has been tested as potential an tiparasitic compounds against P. falciparum cb5r. It is important to remember that, ideally, this drug should possess high specificity for the P. falciparum cb5r but minimal toxicity to the human host. Further research will be needed in order to obtain useful knowledge of the pharmacological properties of this ubiquitous enzym e and draw conclusions on its suitability as an opportune novel drug target. In this project, observing the biochemical and enzy matic behavior of cb5r and cb5 can reveal very important information about the sui tability of these two proteins as drug targets. This investigation will open the doors fo r future studies that will have the

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87 advantage of using these results and apply them to further studies involving the pharmacological characterization of cb5r and cb5 and drug trials.

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88 MATERIALS AND METHODS The first step in this project to characterize a no vel drug target must start with the source of all future assays: a purified protein. T he process that leads to the expression of a protein consists of cloning of the Plasmodium falciparum cb5r and cb5 genes, followed by optimization of the recombinant protein expressi on and further purification. The membrane-bound form of the proteins will be used fo r this study; these are the truncated versions that lack the transmembrane domain since w e are interested in characterizing the soluble form of the proteins. Experimental Design For cloning of the desired genes coding for cb5r and cb5, a biotechnology company (GENEART, Wrzburg, Germany) was employed t o synthesize both genes obtained from the P. falciparum genome database. Importantly, Plasmodium has an ATrich codon bias that complicates heterologous prote in expression in E. coli Therefore, for synthesis of the genes, we designed the P. falciparum cb5r and cb5 using codon optimization for E. coli to obtain optimal protein expression. Both synthe sized genes were subcloned into the vector plasmid vector calle d pTWIN (New England Biolabs). This vector has several features: (a) the expressio n of the fusion gene is under the control of the T7 promoter and is regulated by IPTG due to the presence of a lacI gene and (b) that the expression requires an E. coli host that carries the T7 RNA polymerase gene [ER2566, BL21 (DE3) and derivatives]. Each primer also contains a His6-tag, an Intein protein, and a Chitin Binding Domain (CBD) fragment s for further protein purification.

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89 When the genes have been successfully inserted into the plasmid, the transformation into bacterial cells could proceed. This was where the actual project begun. The bacteria used for this purpose was E. coli BL21 (DE3) RIPL, which is a special strain of bacte ria optimized for heterologous protein expression. Cells were grown in the presence of LB broth and pl ated on Terrific Broth (TB) agar containing 100g/ml ampicillin. Our original genes that were liga ted into plasmids contained an ampicillin-resistant gene that allowed growth of those cells that retained the plasmid. Positive clones were then sequenced to co nfirm that no mutations occurred during the cloning process. Followed transformatio n, minipreparation of bacterial plasmid DNA and digestion of minipreparation by res triction enzyme were performed. Agarose gel electrophoresis was then performed to o btain a DNA gel in which the bands corresponding to the base pairs number of plasmid a nd insert could be observed. Optimization of recombinant protein expression was determined according to temperature (23 C), time of induction (16 hours), and IPTG concentration (0.2, 0.5, 1.0 mM). Cells were sonicated and separated into pelle t and supernatant. This separation also aided in identifying the presence of cb5r and cb5 in either the pellet or the supernatant; since the two proteins are soluble, th ey were expected to be found in larger amounts in the supernatant. The purification process started with the expressi on in E. coli of the inserted plasmid; later on the recombinant protein expressio n was achieved by the addition of IPTG, followed by sonication. Finally, purificatio n was obtained by Chitin Agarose Chromatography and Nickel Column Chromatography.

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90 Methods Vectors with the inserted clones were transformed in E. coli BL21 (DE3) RIPL competent cells and plated on TB agar containing 10 0g/ml ampicillin. Positive clones were selected from previously streaked backup plate s and sent to Moffitt Core Sequencing Laboratory for sequencing. Bacteria wer e grown overnight in 2ml LB broth with 75l/ml of ampicillin; the sample was subsequently was hed (minipreparation) with a series of buffers with the ultimate purpose of lysi ng the cells and cleaning DNA plasmids from proteins and other substances. The following buffers in the following concentrations were added: 300l TENS Lysis buffer – to break the cells apart and release content 150l 3M NaAc – caused proteins that were present in th e released content to precipitate and separate from DNA and forms a prote in precipitate (solid) 900l 100% EtOH – helped precipitate DNA Following precipitation at – 20C for 15 minutes, p ellet precipitate was washed first with 500l 70% EtOH (cleans the plasmids from salts and buff ers) followed by 500l 100% EtOH (dries up DNA and gets rid of water left on DNA from previous wash) DNA gel analysis was performed using Agarose Gel El ectrophoresis to confirm the correct insert size in the pTWIN vector.

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91 Optimization of Protein Expression P 138 P. falciparum cb5 and cb5r in E. coli BL21 (DE3) RIPL cells were grown overnight in 2mL TB-Amp75-chloramphenicol (Cm) 50g/mL at 37C and 250 rpm. A 1:100 dilution of overnight culture was added to a 70mL fresh TB-Amp75 broth containing either 100M riboflavin or 100M ferric citrate; the sample was then grown for 2-2.5 hours at 37 C at 250 rpm until an Optica l Density at 600 nm (OD600) was reached. IPTG was then added to the broths at conce ntrations of 0.2, 0.5, and 1.0mM and grown at either 37C for another 3 hours or overnig ht (16 hours) at room temperature (23C). In order to better localize our proteins, t he cells were disrupted by sonication and centrifuged to separate pellet (P) and supernatant (S/N). SDS-Polyacrylamide Gel Electrophoresis was then performed in 4-20% acrylam ide gels. Protein Purification The cells were harvested by centrifugation at 2500 rpm for 10 minutes at 4 C. The pellet was washed with PBS and the resuspended in lysis bu ffer. Cells were then sonicated and centrifuged at 10000 rpm for 5 minutes. The product is the separation of the sample into pellet and supernatant. Our proteins are soluble th erefore we expect them to be found in the supernatant. The pellet was then washed with PB S to remove all traces of supernatant and spun. Pellet and supernatant are now ready to b e run into a gel. The proteins were further purified by two processe s: Chitin Agarose Chromatography and Nickel (Ni-NTA) Column Chromatog raphy. In Chitin Agarose Chromatography the protein passes through chitin re sin beads which bind the target protein and the intein by the CBD. Subsequently, t he target protein is cleaved by the use

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92 of a thiol-induced cleavage buffer of the intein, a nd the soluble protein of interest is eluted (Fig. 18). In the purification process, a 2 00l chitin bead slurry was spun at 10000 rpm for 30 seconds. The beads were washed with lys is buffer and added to the gel bed. After incubation, beads were washed again with lysi s buffer for five times. An additional wash with lysis buffer containing 40mM DTT was then applied. Centrifugation led to a supernatant (elution 1), then addition of DTT buffe r, incubation for 1 hour at room temperature, and subsequent centrifugation led to e lution 2. DTT buffer was added again and centrifuging led to elution 3. Before being run through a nickel column, the prot ein was bound to the Histidinetag only. The His-tag gave the protein an enhanced affinity that would aid in the purification. Usually the target protein is the on ly protein in a mixture that would have such a strong bond to a long chain of histidine res idues (usually six residues). This tag has high affinity towards nickel. When the protein is run through a resin with an abundance of nickel, the His-tag will bind the nick el and gets retained in the resin while all other proteins are will pass through. Imidazol e would then be added to the resin, which will compete with his-tags for nickel binding since they have similar structure. The protein was then released from the resin in a p rocess called elution. In the Ni-NTA column chromatography, the column was equilibrated with lysis buffer and centrifuged at 2000 rpm for 2 minutes. The lysate from chitin aga rose purification was loaded into the column and allowed to sit for 30 minutes at room te mperature. The sample was then centrifuged again at 2000 rpm for 2 minutes and the column was washed with lysis buffer. Finally the protein was eluted with elutio n buffer and spun as before, leading to a

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93 supernatant (Ni-NTA elution 1). Elution buffer wa s again added and centrifuging the sample led to elution 2. A repeat of the same proc ess led to elution 3. Figure 18: Protein Purification by Chitin Agarose C hromatography Using pTWIN Vector. Fusion of the intein protein to the C-terminus of P. falciparum cb5r target protein and release of the P. falciparum cb5r by thiol-induced cleavage. In this method, the ta rget gene is cloned adjacent to an intein endoprotease (thiol inducible) linked to a chitin b inding domain (CBD) that allows purification by chi tin chromatography. Subsequently, thiol-induced cleavag e of the intein releases the His6-cb5r or His6-cb5 protein that is further purified by standard Ni-NTA column chromatography.

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94 RESULTS The pTWIN vector containing both P. falciparum cb5r and cb5 genes, was transformed into E.coli BL21 (DE3) RIPL cells and confirmed by sequencing. After minipreparation and digestion of minipreparation, a n Agarose Gel Electrophoresis was performed to visualize if the inserts were successf ully digested by restriction enzymes (Fig 19). Figure 19: DNA Gel of Cb5 Gene Coding Insert by Agarose Gel Electrophoresis.

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95 In this DNA gel the first column is the DNA ladder, while the following columns represent four differe nt plasmids that were picked by selecting four differe nt bacterial colonies in TB agar. The top band with the red spot in all four pTWIN columns is the 5426 base pairs vector containing the insert. The bands with the green spot represent the 1234 base pairs insert. Th e cb5 is composed of 432 base pairs that are included in the 1234 base pair insert. Heterologous Expression of P. falciparum Cb5r Protein in E. coli The expression of P. falciparum cb5r could not be achieved during this sevenmonth project. Numerous different experiments were attempted in order to optimize the expression of the protein but none of these succeed ed. One conclusion drawn from our studies and experiments was that Plasmodium cb5r may be toxic to the strain of E. coli used throughout our research. Specifically it may use up or deplete the cells of crucial molecules, such as NADH, that play a role in vital biochemical processes. In the same laboratory, a very similar result was observed in t he expression of cb5r of the pathogenic fungus Cryptococcus neoformans when the same strain of E. coli [ E. coli BL21 (DE3) RIPL] was used for protein expression. Interestingl y, cb5 expression was very abundant while cb5r expression was not obtained. Since expression in the bacterium E. coli has not been successful for Plasmodium cb5r, one possible solution would be the use of a different organism, such as the fungus Pichia pastoris Plans have been made for this alternative future approach in the laboratory. Specifically, as for cb5 expression in E.coli our first attempt of heterologous expression in Plasmodium cb5r was performed by (1) induction at various IPTG concentration (Fig. 20) followed by standard protei n purification steps (Fig. 21). Then a series of different assays were performed using dif ferent procedures with the goal of reaching optimal expression; (2) the addition of de tergents during the purification steps (Fig. 22), (3) growth at different temperatures and for different incubation periods (Fig.

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96 23), (4) growth in different media (Fig. 24), (5) a ddition of alcohol to the growth medium (Fig. 25), and (6) the screening of different bacte rial clones (Fig. 26 & Fig. 27). The results of these different optimization attempts ar e described in detail as follows: 1) Optimization of P. falciparum cb5r protein expression using different concentrations of IPTG (0.2, 0.5, and 1.0 mM). Also shown are the pe llet and supernatant obtained by sonication of the cells (Fig. 20). The samples wer e then purified by standard Chitin Agarose Chromatography and Ni-NTA Column Chromatogr aphy but no significant improvement was obtained; hence, the uninduced and induced samples looked the same (Fig. 21). 2) Optimization of cb5r expression was performed by adding Triton X-100 a nd SDS detergents to the pellet and supernatant in order t o solubilize the content, such as inclusion bodies, from the pellet. The protein may be found in the pellet, therefore by solubilizing the content we may obtain a better yie ld of cb5r, but again, not differences could be observed (Fig. 22). More specifically, pr oteins expressed with bacteria sometimes end up in inclusion bodies that serve as “waste compartment” for the bacteria. These inclusion bodies are usually found as a solid form or as precipitates, therefore the addition of detergent might help solubilize these s tructures and release the proteins (cb5r) into the supernatant fraction. 3) Despite our finding that for most other protein expression experiments the optimal growth conditions were overnight (16 hours) culture at room temperature (23 C), growth

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97 was also performed at 37 C for 2 hours to compare the outcomes. It was thought that cb5r may need a more rapid and sudden expression to pr event bacterial degradation, but no substantial difference was found, and the bands corresponding to the molecular weight of cb5r have the same intensity for the two different gro wth conditions (Fig. 23). 4) In the early stages of optimization of protein e xpression, two different growth media were used (LB broth and Terrific broth), and sample s were selected at different times of incubation (30, 60, and 120 minutes). LB broth is less rich in nutrients than terrific broth, to stimulate growth, hence this medium was chosen a s an alternative to the nutrient-rich Terrific broth. The rationale here is that since c b5r is toxic to E. coli the protein may need a less powerful growth medium. It could also be the case that cb5r needs less time for proper folding, after which it begins degrading Therefore three different expression times were chosen (30, 60, and 120 min). Again, th e two media and the incubation time at which the samples were collect did not affect ex pression of Plasmodium cb5r (Fig. 24). 5) As a next step, two different temperatures were used (16C and 30C) with two growth media (LB and Terrific broth) and with different in cubation times (30, 60, 120, and 180 min). Additionally, 2% ethanol was added to some o f the growth conditions with the rationale that ethanol can substitute the lack of c haperone proteins in E. coli as a prokaryote, which can help facilitate protein foldi ng in eukaryotic cells such as Plasmodium Ethanol can take the role of chaperones and aid in the protein folding/unfolding during cb5r expression in the heterologous bacterial system. Nevertheless, no significant differences in express ion were observed (Fig. 25).

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98 6) In figure 26a and 26b, twelve new clones were pi cked and run on a gel to compare them for protein expression levels with the previou sly picked clone, using Plasmodium cb5 as positive control for optimal protein expression (see next chapter on cb5 expression). Again, no difference was observed bet ween any of the twelve newly picked additional clones when comparing uninduced and indu ced conditions on a protein gel (Fig. 26a and 26b). Molecular Weight of P. falciparum cb5r/Intein2-CBD: 66.17 kDa Figure 20: Optimization of P. falciparum Cb5r Protein Expression: Various IPTG Concentrations. Induction by IPTG at different concentrations (0.2, 0.5, 1.0mM) for 16 hours at 23 C. Also shown are the pellet and supernatant obtained after sonication of the bacteria. nrr

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99 Figure 21: P.falciparum Cb5r Purification Gel: Chitin Agarose and Ni-NTA Chromatography. Standard protein purification with Chitin Agarose a nd Ni-NTA Chromatography was performed with 3 and 2 elutions, respectively. Since no significant resu lts were observed in protein expression by IPTG induction, further purification did not show anythi ng either. Figure 22: Optimization of P. falciparum Cb5r Expression Gel: Addition of Triton X-100 and SDS Detergents. Detergents were added to the pellet at 0.1% concent ration to solubilize the content and “free” any pro tein trapped in inclusion bodies. The overexpressed prot ein may be found in the pellet, therefore by solubl izing the pellet we may extract the protein but no differ ences are visible between the pellet and the supern atant.

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100 Figure 23: Optimization of P. falciparum Cb5r Protein Expression: Growth at Different Conditions (Temperature and Incubation Pe riod). Optimal growth was tested at two different temperat ures and incubation periods (37C for 2 hours and 23C for 16 hours) to see which of these two condit ions positively affected expression. No significant differences were observed. Figure 24: Optimization of P.falciparum Cb5r Expression: LB Broth vs Terrific Broth. The use of more or less rich media can affect the p roper expression of a protein. Since cb5r may be toxic to the bacterial cell, we tried to use an alternative less rich medium (LB broth) to compare it with expr ession in our standard medium (Terrific broth). No signifi cant differences were observed.

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101 Figure 25: Optimization of P. falciparum Cb5r: 16C and EtOH. Growth was also tested at 16C in both LB and Terri fic broth. Furthermore, growth was performed with t he addition of 2% ethanol to the growth medium at 30C Ethanol takes up the role of chaperones and is supposed to help cb5r fold and unfold properly since E. coli being a prokaryote, lacks chaperones In vivo Plasmodium parasites, which are eukaryote organisms, use chap erones to perform such function but, since E. coli does not have chaperones, ethanol is thought to su bstitute chaperones. No significant differences were observed. Figure 26a: P. falciparum Cb5r Protein Expression: Screening of Clones 1 through 6. Six new clones were picked and run on a gel to comp are their protein expression levels with the previo usly induced clone, and cb5 as positive control that shows proper expression w hen compared with the uninduced sample. No significant differences were o bserved.

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102 Figure 26b: P. falciparum Cb5r Protein Expression: Screening of Clones 7 through 12. Six additional clones were picked and run on a gel to compare their protein expression levels with the one of the previously picked clone, using cb5 as positive control that shows proper expression w hen compared with uninduced bacteria. No significant differences were observed. Heterologous Expression of P. falciparum Cb5 Protein in E. coli Optimization of protein expression for Lys44-cb5r-pTWIN and Pro138-cb5pTWIN revealed that the optimal growth conditions a re overnight (16 hours) culture at room temperature (23 C) at 100 rpm after being induced with 0.2mM IPTG. After sonication, Pro138-cb5-pTWIN is located in the cell lysate supernatant (F ig. 27).

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103 Figure 27: Cb5 SDS-Polyacrylamide Gel Electrophoresis. The band that corresponds to cb5 (with His-tag, intein, and CBD still attached) is labeled in red and weighs 44.78 kDa. This gel clearly shows the difference in expression between the uninduced (column 2) and th e 0.2, 0.5, and 1.0mM IPTG induced cb5 in columns 3, 4, and 5 respectively. Also included are the 0.2 and 1.0mM IPTG induced pellet (columns 6 and 8 respecti vvely) and the 0.2 and 1.0mM IPTG induced supernatant (columns 7 and 9 respectively) obtained after sonication. Further purification was achieved by Chitin Agarose Chromatography. This purification breaks the bond between the Intein and the His-tag so that the protein (cb5) is left with only the six histidine residues attached (Fig. 28).

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104 Figure 28: Cb5 Gel after Chitin Agarose Chromatography. This gel shows the difference in molecular weight i n the cb5-Intein2-CBD that was induced by IPTG (column 3) and after sonication in the S/N (column 5), both with red stars, compared with cb5 after successful cleavage of intein and CBD (columns 6, 7 and 8 corresponding to elution 1, 2, and 3 respectively) (black star). We subjected cb5 through an additional purification through Ni-NTA Column Chromatography. Here, the protein was separated fr om the His-tag and it became a completely pure protein. Cb5 final molecular weight was calculated to be 16.94 kDa (Fig. 29).

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105 Figure 29: Cb5 Gel after Complete Purification by Ni-NTA Column Chromatography. Bands in column 9 and 10 (red stars) represent the completely purified cb5.

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106 DISCUSSION Our main objective of expressing a recombinant prot ein generated ambiguous results, one of which was that the expression of cb5 was very successful. The type of vector and the strain of bacteria used in cloning a nd expression of a recombinant protein gave positive outcomes with cb5 and the amount of protein yielded was extremely abundant. On the other hand, the expression of cb5r was very problematic and could not be achieved. Numerous attempts were made to achiev e the expression of cb5r, but none was successful. Several approaches were attempted but none of them worked. Transformation of plasmids into E. coli and protein expression was attempted twice from the beginning; it was thought at first that maybe t he clones that were chosen had not picked up our gene. The gel made after a new trans formation attempt failed to show any differences between the uninduced and IPTG induced clones. In another instance, expression by IPTG induction was aided by ethanol w hich was thought to promote protein expression, unfortunately however, the resu lts did not change, and the protein failed to be expressed. Additionally, the expressi on of cb5r was carried out at different temperatures and with different incubation periods but none of the assays produced satisfactory results. A possible solution to achie ve successful purification is to use a different organism for heterologous expression, suc h as the fungus Pichia pastoris Given the time constraints, further experiments cou ld not be performed and the biochemical and kinetic characterization of cb5r and cb5 would have to be conducted at a later time.

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107 The future production of knock-outs of cb5r and cb5 will help us to identify which amino acids are responsible for the binding with th e molecules with which they interact. This will aid to a more complete and thorough under standing of the biochemical and pharmacological characteristics of these two protei ns. In the context of public health, the identificatio n of new successful drug targets for falciparum malaria will have great implications the first and most important being the possible use of alternative drugs and the consequen t alleviation of the current drug resistance. As it was stated throughout the text, the emergence of resistance represents a huge obstacle in the cure of malaria, and this obvi ously leads to a greater number of deaths. The first goal of public health is to prev ent or at least reduce the number of deaths, therefore the discovery of new drug targets represents one of many methods that leads to the reduction of the burden of malaria wor ldwide. The successful expression of cb5r will certainly be achieved in the near future. In this way, the desired biochemical, kinetic, and pha rmaceutical characterization will be investigated and a possible novel drug target disco vered.

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