IDENTIFICATION OF VACCINE AND DRUG TARGETS AGAINST MALARIA HIRDESH KUMAR

Transcription

1 IDENTIFICATION OF VACCINE AND DRUG TARGETS AGAINST MALARIA HIRDESH KUMAR KUSUMA SCHOOL OF BIOLOGICAL SCIENCES INDIAN INSTITUTE OF TECHNOLOGY DELHI MARCH 2016

2 Indian Institute of Technology Delhi (IITD), New Delhi, 2016

3 IDENTIFICATION OF VACCINE AND DRUG TARGETS AGAINST MALARIA by Hirdesh Kumar Kusuma School of Biological Sciences Submitted In fulfilment of the requirements of the degree of Doctor of Philosophy to the Indian Institute of Technology Delhi March 2016

4 CERTIFICATE This is to certify that the thesis entitled Identification of vaccine and drug targets against malaria, being submitted by Mr. Hirdesh Kumar to the Indian Institute of Technology Delhi for the award of the degree of Doctor of Philosophy is a record of the bonafide research carried out by him, which has been prepared under our supervision and guidance in conformity with rules and regulations of the Indian Institute of Technology Delhi. The results therein have not been submitted in part or full to any other University or Institute for the award of any Degree/Diploma. Dr. James Gomes Professor Kusuma School of Biological Sciences Indian Institute of Technology Delhi New Delhi , India Dr. B Jayaram Professor Department of Chemistry and SCFBio Indian Institute of Technology Delhi New Delhi , India Dr. Friedrich Frischknecht Professor Department of Infectious Disease, Heidelberg Clinic Germany Date: Place: New Delhi i

5 Acknowledgements Foremost, I would like to express my sincere gratitude to my supervisors. I am thankful to Prof James Gomes for his continuous support throughout my PhD carrier, his motivation and regular inputs into the problems. I am indebted to Prof. Frischknecht who helped me in developing the scientific attitude and also for his invaluable suggestions throughout my PhD carrier. I am indeed thankful to Prof. Jayaram for his motivation and time to time discussion. I sincerely thank you all for being the sort of supervisors every student needs - astute, supportive, enthusiastic and inspiring. I would like to express my deep sense of gratitude to the following people for their direct or indirect contribution in my thesis: Professors Aditya Mittal, Archana Chugh, Ashok Kumar Patel, Bishwajit Kundu, Chinmoy Sankar Dey, Seyed E. Hasnain, Tapan Kumar Chaudhuri and Vivekanandan Perumal for their teaching. My friends from Kusuma School of Biological Sciences (KSBS), in particular Ashutosh and Vinay who taught me basic molecular biology techniques and to Aditya and Suhas for their time to time discussions. Dr. C. R. Pillay (National Institute of Malaria Research, New Delhi) who introduced me to the malaria biology, in-vitro blood culture and microscopy of the Plasmodium parasite. Dr. Shikhar Gupta (Procter and Gamble, Singapore) for encouraging me to apply for DAAD scholarship. Dr. Ranajit Shinde (Institute of Microbial Technology, Chandigarh) and Dr. Rajendra Kumar (Umeå University, Sweden) for their invaluable discussions on molecular dynamics simulations. Mahendra Awale (University of Berne) to assist in automated virtual screening and Vivek Kumar (National Institute of Pharmaceutical Education and Research, Mohali) for his all-time support and for valuable discussions. I would like to express my great regards to German Academic Exchange Service (DAAD) for awarding me DAAD scholarship to work in Germany and giving opportunity to convert my ii

6 idea into reality. My sincere thanks to Prof. Frischknecht who allowed me to work in his lab and to test my hypothesis. I am thankful to Goethe institute for exciting German language course. I would also like to thank to Miriam Griesheimer who nicely took care of official documents that made my early days in Heidelberg much easier. In Prof. Frischknecht laboratory, I am extremely thankful to Mirko Singer, Jessica Kehrer and Gunnar Mair who introduced me to advanced molecular parasitology techniques and for the in-depth discussions. A special thanks to Miriam Ester, who is always nice to everyone even towards her mosquitoes. My special thanks also to Dennis Klug and Ross Douglas for critically reviewing my work. My sincere thanks to Kartik for all his support, stimulating discussions, for his company during weekends and sleepless nights when we were working together, and of-course for zussamen essen. I am also thankful to all other lab-mates including Catherine Moreau, Mendi Muthinja, Benjamin Spreng, Katharina Quadt and Konrad Beyer for providing the wonderful working environment. In Mueller s lab, I am thankful to Ann-Kristin Mueller, Kirsten Heiss and Julia Sattler for their time to time discussions and fruitful suggestions. Franziska to try adenoassociated viral production of LISP2. I am also thankful to animal-facility persons who took care of my experimental animals. I am also thankful to Jessica Kehrer, Gunnar Mair, Nikhil Taxak, Julia Sattler for reading my thesis and making necessary corrections. Last but not least, I am thankful to my parents who gave me strength to work. My brother to provide me mental peace to think better and work harder. And to my lovely wife for her eternal love, sound patience, all time support and encouragement, and for having faith in me. Hirdesh Kumar iii

7 Abstract Malaria parasites have become resistant to promising artemisinin-combination therapy (ACT) and leading vaccine candidate RTS,S has also failed to provide long-term protection in humans. The Malaria Eradication Program needs a new generation of antimalarial drugs and effective vaccines against the disease. An ideal antimalarial drug should arrest the parasite in all development stages in humans and thus inhibit the target protein in each of these stages. Similarly if vaccines are developed, these needs to be specific for a certain stage. For example, parasites that lack genes that encode proteins with essential functions during asexual replication or red blood cell invasion, cannot be made or evaluated for their potential as liver stage specific vaccine. Genetically attenuated parasites (GAPs) that lack genes essential for the liver stage of the malaria parasite, and therefore cause developmental arrest, have been developed as live vaccines in rodent malaria models and recently been tested in humans. Combining the available proteomics data from rodent- and human-parasites, I performed a systematic ortholog-based analysis and identified potential vaccine candidates and drug targets. Next, using reverse genetics approach, I studied the role of five such candidates in Plasmodium berghei ANKA: 2 vaccine candidates and 3 drug-targets. The gene of interests were individually deleted in the parasites and the clonal lines were characterized throughout the life cycle (in C57BL/6, NMRI and BALB/c mice; Anopheles stephensi mosquitoes and liver hepatocellular cells). To test first vaccine candidate, I generated P. berghei parasite lines that lacked the entire coding sequence for the late liver stage protein LISP2. The deletion of lisp2 leads to a nearly complete arrest in late liver stage development. The N-terminus of the protein was sufficient to support the complete liver stage development of the parasites. C57BL/6 mice that cleared lisp2(-) parasites, remained protection against further wild-type challenge. iv

8 Then, the second vaccine candidate was tested and ARP encoding gene in the parasites was deleted. Thus developed mutant parasites have a growth-delay during blood stage development and mice infected with such parasites eventually cleared the infection. Although able to form ookinetes, arp(-) parasites did not form oocysts in mosquito midguts and were not transmitted through mosquitoes. Mice given arp(-) parasites were immune to the subsequent challenge with blood stages of P. berghei strain ANKA and P. yoelii YM and to the P. berghei strain ANKA sporozoites. For testing drug-targets, I hypothesized that depletion of house-cleaning enzymes involved in the conversion of toxic metabolites could potentially lead to the arrest of parasite growth because accumulation of these metabolites might kill the parasites. The non-canonical nucleotides like dutp, ditp and dxtp, which are generated during metabolism of canonical nucleotides (trinucleotides of A, T, G and C) incorporate into the nascent DNA leading to DNA break and further to cell death. Through chem-bioinformatics analyses, I predicted three housecleaning enzymes in Plasmodium: deoxyuridine 5 -triphosphate pyrophosphatase (dutpase); inosine 5 -triphosphate pyrophosphatase (ITPase) and NUDIX-domain containing protein (NuDiP). These enzymes detoxify non-canonical nucleotides. Using reverse genetics approach, I tested 3 candidates and found that dutpase was essential for P. berghei blood stage while NuDiP and ITPase were dispensable. v

9 Table of content CERTIFICATE... i Acknowledgements... ii Abstract... iv List of figures... x List of tables... xii Abbreviation... xiii Chapter 1: Introduction Global impact of the disease Complexity of the disease Life cycle of the parasite Clinical features of malaria Malaria complications Cerebral malaria Severe anaemia Diagnosis of malaria Microscopy Rapid Diagnostic Test (RDT) Current antimalarial therapy Artemisinin derivatives Artemisinin combination therapy (ACT) Antimalarial drug resistance Artemisinin resistance Alternatives to artemisinin resistance Vaccine against malaria Pre-erythrocytic vaccine P. berghei: A model rodent parasite vi

10 1.11 Scope of the research Definition of problems and objectives Chapter 2: In silico identification of vaccine and drug targets against malaria Introduction Results and Discussion Search for GAPLS candidates Search for multi-stage drug targets Conclusions Chapter 3: Deletion of lisp2 arrests P. berghei ANKA liver stages in C57BL/6 mice and protects against further infection Introduction Results and Discussion LISP2 is a Plasmodium specific protein with conserved, 6-Cys domain Incomplete deletion of lisp2(-) did not result in a complete arrest Complete disruption of lisp2 results in an almost complete arrest in the liver amino-acid long N-LISP2 is capable to complete liver stage development Immunization with lisp2(-) sporozoites confers dose dependent protection Additive attenuation in malaria parasites lacking two liver specific genes Summary Chapter 4: Plasmodium berghei arp(-) parasites are virulence attenuated blood stages and induce protective immunity against experimental malaria Introduction Results and Discussion ARP is a conserved Plasmodium protein with a dsdna_bind domain PbARP is expressed in all stages ARP(-) parasites form attenuated blood stages in infected mice ARP(-) parasites arrest during mosquito-stage development vii

11 4.2.5 Recovery from ARP(-) infection results in long-lasting malaria immunity Deciphering the role of ARP in the parasite MYST is essential for P. berghei ANKA blood stages Can we predict GAPBS candidates? Summary Chapter 5: Identification of essential house-cleaning enzymes in Plasmodium berghei Introduction Results and Discussion Identification of detoxifying enzymes using chem-bioinformatics analyses Three detoxifying genes are expressed during blood and mosquito stages Failure to delete dutpase in P. berghei Parasites lacking ITPase complete their life-cycle Nudix-domain containing protein (NuDiP) has dispensable role PbdUTPase consists an additional insertional motif Summary Chapter 6: Materials and methods Identification of vaccine targets Ortholog and paralog search in different Plasmodium species Phyletic distribution Percentage identity matrices Identification of multi-stage drug-targets Unique, multi-stage expressed P. falciparum proteins Plasmodium protein lacking similar human protein Search for similar crystal structure Search for essential genes Homology modeling and crystal structure analysis Molecular docking of non-canonical nucleotides (chapter 5) viii

12 6.5 Expression analysis of GOIs Preparation of knockout vectors Transfection vectors Cloning Generation of mutant P. berghei ANKA parasites lacking GOI Ethics statement Animals and parasites Transfection Characterization of mutant parasites across the life cycle Limiting dilution Phenotypic characterization of GOI(-) blood stages Mosquito-stage infection of GOI(-) parasites Infectivity of the mutant sporozoites Recycling of selection marker (Negative selection) Immunization and challenges arp(-) GAPBS lisp2(-) GAS Summary and conclusions Conclusions References Biodata ix

13 List of figures Figure 1.1: Life cycle of Plasmodium falciparum... 4 Figure 2.1: Search space for identification of drug-targets and GAPLS Figure 2.2: Identification of liver stage candidate genes in human parasites Figure 2.3: Percentage identity matrices of 20 shortlisted vaccine candidates Figure 2.4: Identification of multi-stage drug-targets in P. falciparum Figure 3.1: Percentage identity matrices of LISP2 in different Plasmodium species Figure 3.2: LISP2: 6-Cys domain in Plasmodium genus Figure 3.3: Different attempts to delete lisp2 in P. berghei ANKA parasites Figure 3.4: lisp2 deletion strategy in Plasmodium berghei Figure 3.5: Liver stage development of lisp2(-) sporozoites Figure 3.6: Mosquito stage development of lisp2(-) parasites Figure 3.7: lisp2(-) parasites arrest during late liver stages Figure 3.8 Recycling of selection marker Figure 3.9: Complementaiton of lisp2(-) parasites with N-LISP2 encoding sequence Figure 3.10: 493 amino acids of LISP2 rescue the liver stage arrest of lisp2(-) parasites Figure 3.11: C57BL/6 mice immunized with different GAS Figure 4.1: Gene and protein architecture of PbARP Figure 4.2: Analysis of PbARP expression in P. berghei life cycle Figure 4.3: Targeted gene disruption of ARP in P. berghei Figure 4.4: ARP (-) parasites showed delayed growth during blood stage Figure 4.5: ARP depleted parasites form less ookinetes and form no oocyst Figure 4.6: ARP(-) parasites generate protective immunity Figure 4.7: Schematic representation of ARP signaling network Figure 5.1: Docking of non-canonical nucleotides in dutpase and ITPase x

14 Figure 5.2: A, Role of dutpase, ITPase and NuDiP as detoxifying enzymes Figure 5.3: Comparison of active site region of ITPase and dutpase Figure 5.4: Expression analysis of 3 detoxifying genes Figure 5.5: The preparation of parasites expressing GFP-tagged genes Figure 5.6: Expression profile and subcellular localization of three proteins Figure 5.8: Targeted gene disruption of ITPase in P. berghei Figure 5.9: ITPase (-) parasites showed normal growth during blood stage Figure 5.10: ITPase (-) parasites showed form normal mosquito stages Figure 5.11: ITPase(-) parasites are delayed in vivo liver stage development Figure 5.12: Gliding motility of ITPase (-) (KO) and wild-type (WT) parasites Figure 5.13: Liver stage development of (ITPase(-)) parasites Figure 5.14: Chem-Bioinformatics analysis of NuDiP Figure 5.15: Targeted gene disruption of NuDiP in P. berghei Figure 5.16: Characterization of NuDiP(-) parasites Figure 5.17: Sequence alignment of the different dutpases xi

15 List of tables Table 1.1: Distinction of different Plasmodium species based on microscopy... 8 Table 2.1: Final shortlisted 20 liver stage specific GAP candidates in P. falciparum Table 2.2: 43 shortlisted multi-stage drug-targets against malaria Table 3.1: 6-Cys family members in Plasmodium Table 5.1: 24 hydrolase in P. berghei Table 5.2: Residues involved in H-bond interactions in docking studies Table 6.1: Homology modeling of shortlisted proteins Table 6.2: Transfection vectors used to tag genes with GFP encoding sequence Table 6.3: Details of primers used for different cloning reactions Table 6.4: Details of vectors used in different knockout strategies xii

16 Abbreviation 5-FC ᵒC ACT ARP bp BS BSA C C57Bl/6 cdna CQ CSP hdhfr DDT DHFS DMEM DNA dntps ds dutpase ECM E.coli egfp FBT FCS FDA g GA GAP GAPBS GAPLS GAS gdna GFP GOI h HAT i.p. i.v. IFA ITP ITPase irbcs kb kda K/X L 5-fluorocytosine degree celcius artemisinin combination therapy apoptosis related protein base pairs blood stage bovine serum albumin celsius C57 black 6, inbred mouse strain complementary DNA chloroquine circumsporozoite protein human dihydrofolate reductase dichlorodiphenyltrichloroethane dihydrofolate synthase doulbecco s Modified Eagles Medium deoxyribonucleic acid deoxynucleotides double-stranded deoxyuridine-triphosphatase experimental cerebral malaria Escherichia coli enhanced green fluorescent protein fresh blood transfer fetal calf serum food and drug administration unit for centrifugation step glutaraldehyde genetically attenuated parasites blood stage arresting GAP liver stage arresting GAP genetically attenuated sporozoites genomic DNA green fluorescent protein gene of interest hour histone acetyltransferase intraperitoneal intravenous immunofluoroscence assay inosine triphosphate inosine triphosphate (ITP) pyrophosphohydrolase infected red blood cells kilo base kilo daltons ketamine/xylazin liter xiii

17 LAVs live attenuated vaccines lisp2 liver stage specific protein 2 MG midgut min minutes MYST protein under family that include MOZ, Ybf1/Sas3, Sas2 and Tip60 as other members NA numerical aperture NMRI Naval Medical Research Institute, refers to an albino outbred mouse strain nt nucleotide N-LISP2 N-terminus of LISP2 ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction PDCD5 programmed cell death 5 Pb Plasmodium berghei Pf Plasmodium falciparum Py Plasmodium yoelii PFA paraformaldehyde p.i. post infection PM plasma membrane PVM parasitophorous vacuole membrane RDT rapid diagnostic test RPMI Roswell Park Memorial Institute medium RBC red blood cell RT room temperature RTS,S/AS01 central repeat region (R) of P. falciparum circumsporozoite protein (CSP) flanked at both ends with T-cell epitopes (T) of CSP using the hepatitis B surface antigen (S) as a carrier matrix. The additional S stands for its expression in Saccharomyces cerevisiae (S) containing liposomal-based Adjuvant System (AS01) sec seconds sm selection marker ss single-stranded SD standard deviation SG salivary gland U units UTR untranslated region WHO world health organization WT wildtype yfcu a bifunctional protein that combines yeast cytosine deaminase and uridyl 5FC 5 -fluorocytosine xiv

18 Thesis organization This thesis is organized in seven chapters. In the first chapter, the literature survey about malaria and current antimalarial therapy have been described. This is followed by a brief introduction about the definition of problem, motivation and specific objectives of the study. Second chapter focuses on in silico identification of potential vaccine- and drug-targets against the malaria parasites. In next three chapters, I tested two of previously identified vaccinecandidates and three predicted drug-targets. Chapter three involves generation of knockout P. berghei parasites lacking lisp2, a late liver stage specific gene, and evaluation of their vaccine potential. The fourth chapter constitutes the experiments to generate arp(-) parasites and testing of their vaccine potential. In chapter five, a chem-bioinformatics based approach to shortlist three house-cleaning drug-targets, is described. The essentiality of each of these candidates has been evaluated. In the final chapter, material and methods used in the execution of the work required for this research study are discussed. The biodata consisting of published articles, conference proceeding, and abstracts are included at the end of the thesis. xv