THE IMPLICATION OF PREFOLDIN AND ASSOCIATED FACTORS ON THE PLASMODIUM INFECTION OF ANOPHELES GAMBIAE by Purnima Ravisankar A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science Baltimore, Maryland April, 2017 ABSTRACT Anopheles gambiae is the dominant vector species of human malaria, caused by protozoan Plasmodium, in Sub-Saharan Africa. In 2015, 212 million cases of malaria were recorded worldwide and Plasmodium falciparum is the biggest killer of African children aged 1-4 years. The efficacy of existing methods of malaria control are compromised by the rise of drug resistant parasites and insecticide resistant mosquitoes. This work focused on studying the role of certain immune genes found as a part of the interactome of a Plasmodium agonist prefoldin. Through RNAi it was determined that of the three genes (TEP15, LRIM26, enolase) studied, enolase seems to play an agonistic role in the development of Plasmodium falciparum in An. gambiae. Silencing of enolase resulted in a lower oocyst load compared to the control. The co-silencing of enolase and prefoldin, however, did not result in significant decreases of oocyst loads compared to silencing of individual components alone. These results suggest that the two genes may be involved in the same pathway/mechanism of regulating Plasmodium infections in the vector. Furthermore, the specific role of prefoldin subunit-5 protein was evaluated by introducing the recombinantly expressed protein in the vector An. gambiae, and performing P. falciparum infection assays. The introduction of recombinant prefoldin ii subunit-5 did not impact oocyst loads in the midgut under the trialed conditions suggesting that further studies are needed to determine the role of the subunits in Plasmodium infections. Thesis Advisor/ Primary Reader Dr. George Dimopoulos Molecular Microbiology and Immunology Thesis Secondary Reader Dr. Conor McMeniman Molecular Microbiology and Immunology iii ACKNOWLEDGEMENTS First, I would like to thank Dr. George Dimopoulos, for giving me the opportunity to be a part of his lab. I would like to thank him for being a patient mentor, and for all the support he has provided. I have been given the chance to learn and grow in a nurturing environment provided by him and his lab and I am grateful for to him for this opportunity. I would also like extend my gratitude to thank all the past and present members of the Dimopoulos lab: Dr. Seokyoung Kang, Dr. Yuemei Dong, Dr. Nahid Borhani Dizaji, Dr. Yesseinia Anglero-Rodriguez, Dr. Sarah Short, Dr. Jenny Carlson, Dr. Maria Luisa Simoes, Raul Saraiva, Hannah MacLeod, Jingru Fang, and Sarah Van Tol for all the encouragement, support, discussions and advice during my time in the lab. I would especially like to thank Seokyoung and Yuemei for all the expert training and without whom I could not have completed my work successfully. I would like to thank my thesis reader Dr. Conor McMeniman for taking the time to review my thesis, and for his counsel about my future in research. I would like to thank Christopher Kizito from the JHMRI insectary, Dr. Abhai Tripathi and Dr. Godfree Mlambo from the JHMRI Parasite Core Facility for the mosquito rearing and parasite cultures respectively. I wish to thank the faculty, staff and iv my peers in the W. Harry Feinstone Department of Molecular Microbiology and Immunology at the Johns Hopkins Bloomberg School of Public Health. I would like to thank my parents for their unconditional love, support, and for always encouraging me to pursue my dreams. I would like to thank my friends for being there for me through the thick and thin of graduate school and for motivating me to work harder and challenge myself. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………………... ii ACKNOWLEDGEMENTS……………………………………………………………... iv TABLE OF CONTENTS………………………………………………………………... vi LIST OF TABLES……………………………………………………………………… vii LIST OF FIGURES……………………………………………………………………..viii INTRODUCTION……………………………………………………………………….. 1 MATERIALS AND METHODS………………………………....…………………….. 30 RESULTS……...………………………………………………....…………………….. 38 DISCUSSION....………………………...……………………………………………… 54 FUTURE DIRECTIONS………………...………………………………………………60 REFERENCES…………………………………………………………………………. 61 CURRICULUM VITAE………………………………………………………………... 71 vi LIST OF TABLES Table 2.1: RNAi Primers……………………………………………………………….. 36 Table 2.2: Prefoldin Protein Subunit Sequences………………………………………... 38 LIST OF FIGURES Figure 1.1. Reported deaths due to vector-borne diseases worldwide in 2002……………2 Figure 1.2. The projected changes in the incidence of malaria from 2000-2015………... 5 Figure 1.3. The life-cycle of malaria-causing Plasmodium parasites……………………. 9 Figure 1.4. The global distribution of dominant vector species and important vectors of malaria across the globe………………………………………………………………… 49 Figure 1.5: The global map highlighting malaria endemic regions…………………….. 15 Figure 1.6. Innate Immune Responses of Drosophila………………………………….. 17 Figure 1.7. The major immune pathways involved in defense against invading pathogens in the vector Anopheles has been illustrated………………………. 21 Figure 3.1. Silencing assay of TEP15 gene…………………………………………….. 39 Figure 3.2. Silencing assay of LRIM26 gene…………………………………………... 41 Figure 3.3. Silencing assay of enolase gene……………………………………………. 43 vii Figure 3.4. Analysis of plasmid from transformed colonies……………………………. 46 Figure 3.5. The Tris-Gly gel post electrophoresis comprising of the His-tagged prefoldin subunit-5 eluates…………………………………………………. 48 Figure 3.6. Plasmodium infection assay post injection of recombinant prefoldin subunit-5…………………………………………………….. 50 Figure 3.7. Trial-1: Midgut oocyst loads in Plasmodium infection assay post oral ingestion of recombinant prefoldin subunit-5 (0.25mg/mL concentration) ………...….. 52 Figure 3.8 Trial-2: Midgut oocyst loads in Plasmodium infection assay post oral ingestion of recombinant prefoldin subunit-5 (0.25mg/mL concentration) ...………….. 54 Figure 3.9 Midgut oocyst loads in Plasmodium infection assay post oral ingestion of recombinant prefoldin subunit-5 (0.5mg/mL concentration) ……………... 56 viii Chapter 1: Introduction The Global Menace: Malaria Malaria is caused by the protozoan parasites belonging to the Plasmodium genus and is transmitted via the bite of infected female mosquitoes belonging to the genus Anopheles (2). In 2015, as many as 212 million malaria cases were recorded worldwide, and the majority of people who succumbed to the disease were children in the African continent (3). The lack of an effective vaccine against Plasmodium, coupled with the development of drug resistant parasites as well as insecticide resistant mosquitoes, corroborate the need for the development of novel strategies for malaria prevention and eradication. The current methods being employed to control malaria include targeting the parasite in the human host using therapeutic drugs and the Anopheles mosquitoes using insecticide treated bed nets or indoor residual spraying with insecticides. While they have shown some degree of efficacy, challenges such as insecticide and drug resistance continue to impede control efforts in several malaria endemic regions. 1 The Dimopoulos group’s research focuses on the study of vector-pathogen interactions to develop novel intervention strategies based on blocking the pathogen in the mosquito. This thesis focuses on determining the role of selected immune genes in the lifecycle of the malarial parasite Plasmodium falciparum in Anopheles gambiae, the primary vector in Africa. The following section provides the background and rationale for this work. Figure 1.1. Reported deaths due to vector-borne diseases worldwide in 2002. [13] 2 History and Geographical Distribution of Malaria The malaria parasite was first discovered in the blood of infected patients by surgeon Charles Lavern. Although this discovery was made only in 1880, malaria has been plaguing humans for millennia, being mentioned in Indian Vedic texts dating back 1500-800 BC (1). The notion that malaria, Italian translation for “bad air”, could be associated with miasmas rising from swamps persisted for 2500 years prior to the discovery of the parasites in an infected patient’s blood (23) (24). The identification of mosquitoes as vectors of the disease is credited to Patrick Manson, and his work in India was furthered by Ronald Ross, who observed oocysts in the anopheline vector which had fed on an infected patient (24). The distribution of malaria depends on environmental factors such as temperature, humidity, and rainfall. The transmission cycle is sustained in the regions that support the breeding of the vector Anopheles (5). The heaviest burden of the disease is seen in tropical and subtropical regions, but within these regions transmission varies depending on altitude and seasonal factors (5). The high prevalence of malaria in Africa could be attributed to the frequency of feeding and highly anthropophilic behavior exhibited by the Anopheles mosquito (4). Sub-Saharan Africa bears the highest malaria burden and mortality, and P. 3 falciparum is the biggest killer of African children aged 1-4 years (9). While children are primarily affected in regions with a high malaria burden, the population at risk of malarial infection in regions with lower transmission settings, such as South America and Southeast Asia, are adults (9). According to the WHO report, malaria transmission is on-going in 91 countries, a substantial burden of which lies in Sub-Saharan African countries (2). Although