The Implication of Prefoldin and Associated Factors On

Home , Prefoldin

THE IMPLICATION OF PREFOLDIN AND ASSOCIATED

FACTORS ON THE PLASMODIUM INFECTION OF ANOPHELES

GAMBIAE

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

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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.

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

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

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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

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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.

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]

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 malaria is a deadly menace, several countries have utilized various malaria control strategies successfully, Sri Lanka being the latest to join that list (6). Despite several measures taken throughout the world, there are still many countries that are yet to be certified malaria free (6). The current methods employed to control malaria transmission, including vector control and therapeutic drugs to eliminate infection, have certainly helped in reducing worldwide malaria burden, as evidenced by the 37% reduction in the incidence of malaria between 2000 and 2015 (2). Although insecticides and artemisinin-based combination therapies have contributed largely to lower the economic burden caused by malaria, growing challenges such as drug resistance and insecticide resistance promote the need for a molecular entomology-based approach to eliminate malaria (2).

Figure 1.2. The projected changes in the incidence of malaria from 2000-2015, by country, are shown in the above. [22]

Malaria

Malaria in human beings can be caused by one of the five species belonging to the protozoan parasite genus Plasmodium: P. falciparum, P. ovale, P. vivax, P. malariae and

P. knowlesi; however, the majority of infections are a result of infection with P. falciparum or P. vivax (9).

The most lethal human infections are caused by P. falciparum due to the ability of this species to sequester in vital organs, including the brain (7). P. knowlesi (a primate parasite) infections in humans are quite rare but it does represent a threat to travelers who visit regions with high primate-vector densities. Infections with P. vivax and P. ovale are not as rare and the hypnozoite stage of these two parasites can remain dormant and cause relapses of malaria that could result in significant morbidity (8).

Although children in high transmission settings, especially in African countries, are exposed frequently to the parasites they are unable to acquire resistance as easily as they acquire resistance to viral infections such as measles and rubella (10). The ability to develop immunity to the malarial parasite takes several years, thus children who have been exposed to infectious bites are still susceptible to episodes of febrile malaria albeit non-severe (10).

Malaria Life Cycle

The bite of an infected female Anopheles results in the introduction of the

Plasmodium sporozoites into the host. In the phase of tissue schizogony, the motile sporozoites enter the bloodstream and navigate their way to the liver. The sporozoites invade the hepatocytes, and reproduce asexually to give rise to thousands of merozoites during the phase of exo-erythrocytic schizogony, each of which are capable of invading red blood cells. The invasion of erythrocytes, known as the erythrocytic schizogony, is a complex process that involves initial recognition and attachment of the merozoite to the erythrocyte membrane, reorientation and junction formation between the apical end of the merozoite, and formation of the parasitophorous vacuole. In the invaded erythrocyte, schizonts are produced as a result of multiple rounds of nuclear division followed by cytokinesis. The schizonts, comprising of merozoites, are released from the infected erythrocytes and subsequently invade uninfected erythrocytes. The rise in body temperature is correlated with the release of schizonts from infected erythrocytes, and the other accompanying symptoms occur due to the release of tumor necrosis factors and other cytokines. Under certain conditions, a small portion of the merozoites differentiate into microgametocytes and macrogametocytes, which are essential for completing the next phase of the parasite’s life cycle in the mosquito vector (12). The manifestation of the

7 clinical symptoms occurs when the parasite multiplies within erythrocytes in the human host. The inflammatory response from the host towards the blood stage parasite results in flu-like symptoms (11). The sequestration of the infected erythrocytes coupled with a high parasite burden result in severe malaria (11). The life-threatening malarial infections are characterized by symptoms such as severe anemia, difficulty breathing, multi-organ failure and coma and may result in death (11). The parasite is thought to benefit from the sequestration of infected erythrocytes since they can avoid the host’s normal splenic mechanism which is involved in the removal of aged and damaged erythrocytes (11).

The transmission cycle continues when a female Anopheles takes a bloodmeal from an infected individual. In the mosquito’s midgut, the gametocytes fuse to form the zygote. The zygote differentiates into a motile ookinete, which penetrates the midgut epithelium and develops into an oocyst. Within the oocyst, sporozoites develop because of sporogony, and the rupture of the mature oocyst results in the translocation of sporozoites toward the salivary gland. The sporozoites are found in the mosquito’s salivary gland approximately 10-18 days post the consumption of an infectious bloodmeal. The life-cycle of the Plasmodium parasite continues once again with the infected female biting a susceptible human host (12).

Figure 1.3. The life-cycle of malaria-causing Plasmodium parasites. [48]

Malaria Vectors

The genus Anopheles comprises of 465 formally recognized species and about 50 unnamed members of species complexes. Not all Anopheles species are permissive to the

Plasmodium parasite (17) (25). Only 70 species have the ability to transmit human malaria, proving that only certain Plasmodium-Anopheles combinations can support the malaria transmission cycle (17) (25). The study of vector biology enables a better understanding of the difference in permissiveness/ refractoriness exhibited by the different Anopheles species, while understanding local vector ecology can aid in mosquito control efforts (25).

Dominant vector species/ species complexes (DVS) is a term which has been coined to identify the 41 species which are a major public health concern due to their ability tendency to transmit malaria (17).

Anopheles species differ in their feeding as well as resting preferences. The vectors are usually anthropophilic, with a high preference for human hosts, while others may sustain on the blood of domestic animals as well as human beings. Endophilic vectors are characterized by their preference to rest indoors after a bloodmeal while exophilic vectors prefer resting outdoors post blood-feeding. Endophagic vectors bite the humans indoors while exophagic vectors tend to feed on humans outdoors (21).

The dominant vector species/ species complexes vary across the globe and differ

10 on a country-by-country basis. Within some countries, the dominant vector species/ species complexes can even vary regionally, thus requiring detailed information and mapping of predominant species/ species complexes to assess entomological risk. In the case of Sub-Saharan Africa, the lack of dominance of a single Anopheles species over others led to the merging of predictive maps for An. gambiae, An. arabiensis, An. funestus in order represent their equal dominance across the continent (17).

Although there are advances in mapping the distribution and intensity of malaria transmission, there is still a need to demarcate the various dominant Anopheles species present in the different parts of the world, in order to design strategic evidence-based malaria control programs (18). The lack of knowledge on the basic biology of local anophelines frequently results in ill-informed use of insecticides, bednets and indoor residual spraying. For example, An. arabiensis, identified as one of the dominant vector species in some areas, is exophilic and exophagic, and thus targeting this vector’s density using indoor-based insecticide treatments would be inefficient. However, the malaria endemic regions with An. gambiae as a dominant vector species would benefit highly from using indoor residual spraying or the distribution of long-lasting insecticide treated bed nets, and could significantly reduce local malaria transmission (19,20,21).

Figure 1.4. The global distribution of dominant vector species and important vectors of malaria across the globe [49]

Malaria Elimination

Despite several attempts and concerted efforts to eliminate malaria, the disease continues to plague hundreds of thousands of people across the world. The measures utilized to control malaria incidence predominantly involve vector control (14). In malaria endemic regions with a heavy burden, the conventional method of vector control involves the use chemicals to break the transmission cycles and reduce vector densities sufficiently to prevent disease incidence. The other strategies involved in vector control may be classified as biological, environmental or personal protection-based methods (13).

Chemical methods of control involve the use of insecticides and larvicides. An example of an extensively used chemical insecticide is dichlorodiphenyltrichloroethane

(DDT) which was utilized in the Global Eradication Program of Malaria in 1955, and coordinated and supported by WHO (15). However, DDT like several insecticidal compounds, is carcinogenic and poses various risks not only for humans but for also other species of ecosystems (15). The widespread use of insecticides such as DDT against mosquitoes resulted in bioaccumulation of this toxic chemical (13). Another major drawback is the increased spread of mosquito resistance (16).

Currently, there is a need for the development of integrated vector management in order to maximize the disease-control benefit in a cost-effective manner and with a

13 minimal environmental impact. The need for an integrated vector management stems from the experience of utilizing chemicals for vector control and therapeutic drugs which resulted in insecticide and drug resistance.

An integrated approach which requires an understanding of vector ecology as well as local patterns of disease transmission would be better suited to enable malaria elimination whilst protecting the environment. Integrated vector management would involve the use of environmental management and biological controls rather than just utilizing a single approach such as insecticides. Environmental management refers to the modification/removal of the vector’s breeding grounds to stop larval development as well as human-vector contact. Biological controls, such as bacterial larvicides and larvivorous fish, insects and crustaceans, target and kill mosquito larvae (13). Metazoan predators such as the mosquitofish Gambusia, the carnivorous mosquito Toxorhynchites and

Mesocyclops copepods are currently being utilized in studies aimed at diminishing mosquito larval populations. However, the application of biological agents in controlling larval populations suffers from major drawbacks such as the inefficiency during periods of heavy rainfall or tidal intrusion when larger amounts of larvae hatch apart from the delayed increase in the population size of these agents (22). The transient habitats of

Anopheles such as natural pools and puddles created during rains presents another obstacle to the use of biological control agents in larval control (69). Despite these

14 challenges, biological control methods could be used to control and eliminate vector densities without the drawback of the negative impact on the ecosystem that accompany the use of chemical insecticides (13).

Figure 1.5: The global map highlighting malaria endemic regions between 2000-2016.

[50]

Insect Immune System

Insects, like all other organisms, are in contact with their environmental surroundings including exposure to pathogens. Drosophila has been used as a model to study insect immunity (26). The first line of defense in insects is similar to that of mammals; epithelial barriers are present under the cuticle, and around trachea, gut, salivary glands and genital tracts (26, 28). Insects erect a second barrier in the form of clots to entrap the pathogen that limits loss of hemolymph (26). Insects rely on their innate immune system to overcome infections by pathogens, that cross the first line of defense, which comprises of the cellular and humoral immune responses (27) (28).

The cellular response is mediated by circulating blood cells known as hemocytes, which aid in phagocytosis or encapsulation of the invading pathogens (27). Hemocytes differentiate into plasmocytes that are responsible for phagocytosis of microbes and into lamellocytes which encapsulate larger pathogens such as parasites (28). A third class of hemocytes known as crystal cells contain the enzyme phenoloxidase which is involved in the formation of melanin which is deposited around parasites and wounded tissue (27). The humoral immune response is elicited by the presence of pathogens and results in production of antimicrobial peptides and other effectors in the hemolymph. The antimicrobial peptides are secreted by a variety of cells into the insect’s body cavity and act against Gram-negative bacteria, Gram-positive bacteria as well as fungi (27, 28).

Figure 1.6. Innate Immune Responses of Drosophila: (A) Posterior region of a third instar larva showing the cuticle and the trachea acting as a physical barrier against infections. (B) A dead and melanized crystal cell phagocytosed by a plasmatocyte. (C)

Encapsulation of an egg of a Drosophila parasite. Cells surrounding the egg are lamellocytes. (D) Clot formation occurs during wound healing. (E) Crystal cells in contact with the larval cuticle. Melanization occurs in response to intruding pathogens or parasites and is also observed during wound healing (F) Humoral immune reaction:

Antimicrobial peptides are released from the fat body into the hemolymph. [28]

Insect Immune Pathways

The innate immune response towards pathogens involves the production of antimicrobial peptides and other effector molecules (30). The mosquito antimicrobial peptides can be broadly classified into cecropins, defensins, attacin and gambicin (37). The inducible expression of antimicrobial peptides is regulated by two independent pathways; the Toll and immune deficiency (IMD) signaling pathways (29,30). The mutation or loss of the genes involved in the two pathways have been shown to render Drosophila susceptible to bacterial and fungal infections (30).

The transmembrane Toll receptor was initially identified to be involved in dorsal- ventral embryonic development in Drosophila (31,35). The pathway is activated in response to gram-positive bacteria and fungal infections (30). The IMD pathway regulates the immune response to infection by gram-negative bacteria (29-32). These pathways comprise distinct signaling molecules, however, they are initiated by pattern recognition receptors

(PRRs) and result in the activation of NF-kB transcription factors (29,30). In Drosophila, the activation of the Toll pathway results in the activation of NF-kB homologs called

Dorsal and Dif that aid the antifungal response (29). The infection of the host by either fungal or bacterial pathogens results in the cleavage of the host protein Spätzle that serves as a ligand for the Toll receptor and results in the induction of antimicrobial peptides

18 such as Drosomycin and Immune induced molecule 1 (30). The presence of lipopolysaccharide (LPS) activates the IMD pathway through interaction with unidentified PRRs, and leads to the cleavage of the protein Relish. The NF-kB homolog

Relish activates the expression of the genes encoding the antimicrobial peptide Diptericin among others. The antimicrobial peptide Cecropin can be activated by either the IMD or

Toll pathway, and it is believed to be involved in the antibacterial as well as antifungal response (29). The IMD and Toll pathways are involved in anti-Plasmodium defense to varying degrees depending on parasite species. A notable difference in the activation of the IMD and Toll pathway in anti-Plasmodium defense is that Toll pathway suppresses infection with P. berghei while IMD pathway is most effective against P. falciparum infection (67,68).

Infection with pathogens could also lead to the activation of two other signaling pathways, namely, the JAK/STAT and JNK pathways (29). Recently, the JNK signaling pathway has been shown to be important in limiting the Plasmodium infection of the

African malaria vector An. gambiae. The basal expression of thioester-containing protein

(TEP-1) and fibrinogen-related protein 9 (FBN9), key components of the complement- like system produced by hemocytes, is regulated by the JNK pathway. The pathway seems to be involved in limiting Plasmodium survival by initiating induction of enzymes that mediate midgut nitration in response to ookinete invasion (35).

The JAK/STAT pathway comprises of the receptor Domeless (DOME), Janus

Kinase (JAK) and the STAT transcription factor. The binding of cytokine ligand unpaired

(UPD) to the receptor DOME, post infection, induces the phosphorylation of the receptor via the associated JAK tyrosine kinase (32,33). The STAT transcription factor is then accumulated post phosphorylation and dimerization in the nucleus and subsequently results in activation of target genes, a few of which are antimicrobial peptides also expressed in the Toll and IMD pathways (33,35). The JAK/STAT pathway differs from the

IMD and Toll pathways in the targeted stages of parasite development; the former targets early oocyst while the latter decreases ookinete survival. The JAK/STAT pathway functions in a TEP1- independent manner but the Toll and IMD pathways rely on TEP1- mediated lysis of the ookinetes that come in contact with the hemolymph (35). The

JAK/STAT pathway is negatively regulated by the repressor proteins SOCS and PIAS

(36).

Figure 1.7. The major immune pathways involved in defense against invading pathogens in the vector Anopheles has been illustrated. [37]

Anopheles resistance to Plasmodium infection

The Plasmodium gametocytes fuse together in the midgut of the female vector to form the motile ookinete. In order to proceed, the ookinete needs to invade the midgut epithelium and traverse it to reach the basal side prior to developing into oocysts (37). The invasion of the midgut epithelium triggers the mosquito’s immune response when PRRs recognize and bind to PAMPs. The genes upregulated as a result of the immune response varies based on the vector/parasite species combination (37, 38). The invasion of the mosquito midgut by the different Plasmodium species induces certain common immune responses as well as species-specific responses. The survival of ookinetes in the midgut epithelium is influenced by the presence/ absence of parasite agonists and antagonists (37).

The antimicrobial peptide gambicin was first characterized to exhibit antiplasmodial activity against the ookinete stage of the parasite (39). The silencing of Caspar, a suppressor of the IMD pathway, leads to refractoriness to P. falciparum in An. gambiae,

An. stephensi, An. albimanus (37, 40). The silencing of the suppressor of the Toll pathway

Cactus results in resistance toward the rodent malarial parasite P. berghei (37).

The Imd pathway regulates the expression of anti-Plasmodium immune effectors such as APL1, TEP1, LRRD7, FBN9 and LRIM1 (37). The thioester-containing proteins belong to the leucine-rich repeat immune gene family (37.38). The silencing of TEP1 and

LRIM1 through RNAi led to significant increases in Plasmodium oocyst numbers compared to controls, thus indicating that these two factors play an important role as antagonists of Plasmodium (37,41,42). Initially identified for its complement like activity, which is similar to complement factor C3 found in mammals, TEP1 has since then been characterized for its role in the phagocytosis of bacterial pathogen and melanization or lysis of the ookinete stage of Plasmodium (37,41). TEP-1 is soluble in the haemolymph, and requires the aid of scaffolding proteins leucine-rich repeat immune protein 1 (LRIM1) and

Anopheles Plasmodium responsive leucine-rich protein (APL1) to efficiently bind to the surface of the ookinete and prevent self- recognition (37). While silencing of TEP1 and other leucine-rich repeat genes seems to result in disappearance of active TEP-1 in the haemolymph, thereby limiting parasite killing, the exact mechanism of parasite elimination is unknown (37). The other TEPs characterized so far include TEP2, TEP3, and TEP6, and have been associated in the phagocytosis of E. coli, S. aureus, and C. albicans respectively in cultured S2 cells of D. melanogaster (43). Certain members TEP family of genes in An. gambiae as well several other leucine-rich repeat (LRR) genes are known to interact as part of protein complexes to yield protective functions against the malarial parasite (38).

Molecular Co-Chaperone Prefoldin

The proper folding of proteins in a cell requires the action of a group of proteins known as molecular chaperones (44-47). Molecular chaperones therefore have a direct impact on proper cell development and maintenance (46). Chaperonins, belong to this class of proteins and are found in archaea as well as eukaryotes but not in eubacteria (44,45).

Chaperonins can be broadly classified into two groups; the GroEL-GroES folding system and thermosome-CCT (cytosolic chaperonin containing the peptide tcp1 [tail-less complex polypeptide 1]) system (46). CCT is an ATP-dependent class-II chaperonin which aids in the proper folding of actin, α- tubulin, and β-tubulin with the help of co-chaperone prefoldin (PFDN) (44-46). Prefoldin comprises of two α-like subunits, namely PFDN3 and

PFDN5 and four β-like subunits which are PFDN-1, 2, 4 and 6 (46). Together the subunits form a jellyfish-like structure with coiled-coil tentacles which play an important role in substrate specificity in binding to non-native target proteins (46). The hexameric prefoldin protein complex binds to the unfolded proteins, co-translationally, and aids in stabilizing the actin and tubulin subunits forming a binary complex prior to interacting with CCT to form the ternary complex (44,46). The prefoldin protein requires all the six subunits to be fully functional and thereby aid in the folding of actin and tubulin to form microfilaments and microtubules (44-47). Actin and tubulin proteins are important for cell division,

24 motility, molecular transport and cytoskeletal stability, as well as signal transduction (46).

The deletion of prefoldin genes in the yeast Saccharomyces cerevisiae results in the inability of the CCT chaperonin pathway to fold the actin and tubulin proteins, post- translation, thus causing impaired cytoskeletal functions (44, 46). Protein folding increases by a 5-fold in the presence of prefoldin as the co-chaperone prevents the release of non- native proteins from the CCT complex (46,47). Mutagenesis of the prefoldin subunit-3 in D. melanogaster led to a loss of integrity of the spindle structure and function, and the deficient cells exhibited tubulin instability (47). In D. melanogaster and C. elegans the mutation/deletion of the prefoldin genes caused embryonic lethality (44). Murine models with a missense mutation in prefoldin subunit-5 exhibited neuronal defects such as progressive neurodegeneration and hydrocephalus, and rendered male mice infertile (46).

Prefoldin and Plasmodium Infection in An. Gambiae

Earlier studies have shown that among the several putative An. gambiae immune genes induced by Plasmodium infection, is the leucine-rich repeat domain protein-7

(LRRD7) gene. RNAi-mediated silencing of LRRD7 resulted in an increase in

Plasmodium oocyst numbers compared to the control mosquitoes injected with green fluorescent protein (GFP) dsRNA (42). Prefoldin subunit-6 (PFDN6) was identified as an interacting partner of LRRD7 using a yeast two-hybrid screen (Dimopoulos laboratory, unpublished data). The screen relies on interaction between the known protein referred to as the bait and the other proteins of interest, called the prey to facilitate the expression of a reporter gene which further aids in differentiating interacting proteins from the ones that do not (52, 65).

The subsequent testing of PFDN6 for its possible its role in P. falciparum infection, revealed that silencing of PFDN6 resulted in a lower oocyst number compared to control (42,52). Thus, LRRD7 was identified to be an antagonist of Plasmodium while

PFDN6 was an agonist.

Further investigation revealed that inhibition of prefoldin subunit-6 reduces the

Plasmodium oocyst number in various Anopheles vectors such as An. gambiae, An. dirus,

An. stephensi (Dimopoulos laboratory, unpublished data). The interactome of prefoldin

26 subunit-6 was then elucidated via co-immunoprecipitation assays and protein sequencing

(Dimopoulos laboratory, unpublished data). The prefoldin interactome comprised of cytoskeletal genes such as actin, tubulin that could be attributed to the co-chaperone activity of prefoldin in the folding of actin and tubulin proteins. Immune genes TEP15,

LRIM26 and enolase were also a part of the interactome (52).

Thioester-containing protein 15, transcribed from the TEP15 gene, has been shown to possess endopeptidase inhibitor activity. The gene belongs to the family of thioester-containing proteins (TEP), previous studies have shown that expression of this family of genes is upregulated during infection with Plasmodium (51). Leucine-rich immune protein 26 gene belong to the family of leucine-rich repeat immune proteins

(LRIMs), which have been characterized as putative innate immunity receptors (63,64). A few members of this family of genes have been shown to play a role in immune response against Plasmodium, while the function others such as LRIM26 are unknown. The enzyme enolase, also known as phosphopyruvate hydratase, catalyses the conversion of

2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway (53,54). The gene, identified for its interaction with prefoldin subunit-6, has not been implicated previously in the modulation of P. falciparum infection.

Thesis Objectives

The aims of this thesis were to study the role of selected An. gambiae immune genes that were identified as putative interacting partners in the prefoldin subunit-6 interactome and investigate the effect of specific prefoldin subunit supplementation on the P. falciparum infection of An. gambiae. Here, I propose three specific objectives:

Objective 1: To investigate the anti-Plasmodium role of genes which associate with the novel Plasmodium agonist Prefoldin

Objective 2: To clone and express subunit-1 and subunit-5 of the prefoldin protein complex

Objective 3: To investigate the influence of recombinant Prefoldin protein subunit-1 and subunit-5 on P. falciparum infection

CHAPTER 2: Materials and Methods

Mosquito Rearing

An. gambiae larvae were reared on cat food pellets and ground fish food. The adult mosquitoes were maintained on a 10% sucrose solution at 27 0C and 80% humidity with a 12-hr light/dark cycle prior to being subjected to experimental manipulations.

cDNA Preparation and Quantification

The implementation of RNAi-based gene knockdown requires preparation and purification of dsRNA that targets the gene of interest (66). In order to produce template cDNA for dsRNA synthesis, RNA was extracted from female An. gambiae using the

Qiagen RNeasy kit. Template cDNA was synthesized from An. gambiae total RNA using

Promega M-MLV reverse transcriptase kit.

Gene Silencing by RNA interference

An. gambiae genes targeted for RNAi knockdown via adult intrathoracic inoculation (66) included thioester-containing protein-15 (TEP15-VectorBase Annotation

AGAP008364), leucine-rich immune protein-26 (LRIM26-VectorBase Annotation

AGAP005744), enolase (VectorBase Annotation AGAP007827), and prefoldin subunit-6

(PFDN6- VectorBase Annotation AGAP012235). Template cDNA was utilized to obtain the polymerase chain reaction amplified gene fragments for TEP15, LRIM26, enolase, and PFDN6 using the primers mentioned in Table 2.1. The dsRNA was then produced using the HiScribeTM T7 In Vitro Transcription Kit with approximately 2μg of the PCR amplified product for each target gene. The control green fluorescent protein (GFP) dsRNA was produced using a plasmid containing the gene insert as template.

Approximately 69ng of the test and control dsRNA were injected into the thorax of cold anesthetized (3-4) day old female An. gambiae using a nano-injector.

Plasmodium falciparum Infection Assays

An. gambiae, three days post injection, were starved for approximately 2-3 hours prior to blood-feeding. Starved mosquitoes were fed on a combination of serum, blood, and P. falciparum NF54 gametocytes culture. Unfed mosquitoes were then separated from the fed mosquitoes. Fed mosquitoes were maintained on a 10% sucrose solution for

7-8 days post feeding, and were then dissected. Mosquito midguts were stained with a

0.2% mercurochrome dye to visualize oocysts. Oocyst numbers per midgut were determined through light-contrast microscopy. The median number of oocysts per midgut were calculated for each tested gene as well as for the control mosquitoes.

Statistical Analysis

Statistical analysis was performed using the Graphpad Prism software, version

5.0. The Mann-Whitney test was used to assess the significance of the difference between oocyst loads between the various genes tested. All tests considered significance at 95% confidence.

Cloning of Prefoldin Subunits

The prefoldin subunit-1 (PFDN1- VectorBase Annotation AGAP010212) and subunit-5 (PFDN5- VectorBase Annotation AGAP003416) genes were amplified using specific primers (Table 2.2) and cDNA from An. gambiae as a template, utilizing the

Platinum Pfx DNA polymerase kit from Thermo Fisher Scientific. The amplified gene product was then purified with QIAquick PCR purification kit from Qiagen. The vector pET28 and the amplified gene product were separately subjected to restriction digestion with BamHI (HF) and NotI enzymes, obtained commercially from New England Biolabs, in succession. The digested fragments of the pET28 vector and the prefoldin subunit genes were ligated with the help of T4 DNA ligase enzyme. Competent BL-21 E. coli bacterial cells were transformed using the ligated vector-gene product. The bacteria were then plated on a kanamycin LB agar plate, the colonies which not contain the pET28 vector could not grow on the kanamycin containing agar plate due to the lack of kanamycin resistance gene, present in the plasmid. The individual white colonies were further tested to determine if they had been successfully transformed with the vector containing the respective gene inserts. The Plasmid Miniprep Kit from Qiagen was then used to isolate the plasmids from the bacterial cultures. Furthermore, the plasmids were digested with the restriction endonucleases BamHI and NotI and visualized with agarose

32 gel electrophoresis. The fragments thus obtained were sent to the JHMI Synthesis and

Sequencing facility for gene sequencing. The sequencing data in combination with the agarose gel electrophoresis patterns aided in determining whether the prefoldin subunit genes had integrated with the vector pET28.

Protein Expression and Purification

An overnight 5mL broth culture of the transformed bacteria was used to inoculate a larger 100mL LB broth culture. The bacteria were allowed to grow until an optical density equivalent to OD 1 was obtained and induced with 1mM IPTG in a ratio of 1:100 for 24 hours. The bacterial pellet was separated from the broth using centrifugation. The bacteria expressing prefoldin subunit-5 were lysed under native conditions and the recombinant protein subunit was extracted from the cell pellet with the help of commercially obtained GE Ni sepharose. The protein bound Ni sepharose fractions were washed with STE buffer and eluted with His-buffer. The eluted fractions were visualized post electrophoresis in NuPage Tris-Glycine gel and stained with coomassie brilliant blue. The prefoldin subunit-1 expressing transformed colonies were lysed under native as well as denaturing conditions to extract and purify the protein. The protocol used in the former case was similar to that of prefoldin subunit-5. The lysates for prefoldin subunit-1 were also obtained under denaturing conditions using the protocol described in the

QiaExpressionist handbook. The protein containing lysates were treated with Ni sepharose beads prior to being purified and eluted. The lysates and eluted fractions were subjected to gel electrophoresis and stained with coomassie brilliant blue.

Effect of Recombinant Prefoldin Protein Subunit-5 on Plasmodium infection

The recombinant prefoldin subunit-5, at concentrations of 0.25mg/mL and

0.5mg/mL, was injected into the thorax of cold anesthetized 3-4 days old female An. gambiae using a nano-injector. An. gambiae were fed on a combination of serum, blood, and P. falciparum NF54 gametocytes culture 3 days post-injection. A separate cohort of

(3-4) day old female An. gambiae were co-fed the recombinant prefoldin subunit-5, at concentrations of 0.25mg/mL and 0.5mg/mL, and P. falciparum NF54 gametocytes culture respectively. The fed mosquitoes were separated from the unfed mosquitoes and maintained on a 10% sucrose solution for 7-8 days post feeding and were then dissected.

The mosquito midguts thus obtained were stained with a 0.2% mercurochrome dye. The oocyst numbers per midgut were determined through light-contrast microscopy. The median number of oocysts per midgut were calculated for each prefoldin subunit and also for the control mosquitoes injected or cofed with bovine serum albumin protein at concentrations of 0.25mg/mL and 0.5mg/mL.

Table 2.1 RNAi Primers

All RNAi primers contain the T7 promoter sequence. 36

Table 2.2 Prefoldin Protein Subunit Primer Sequences

The primer sequences contain the BamHI restriction sequence (GGATCC) and the NotI restriction site (GCGGCCGC) in order to facilitate restriction digestion by these two restriction endonucleases.

CHAPTER 3: Results

RNAi Mediated Gene Silencing

RNAi mediated gene silencing of TEP15, LRIM26 and enolase was performed by intra-thoracically injecting dsRNA synthesized against each respective gene into 3-4- day old female An. gambiae. After an infectious blood meal, the median P. falciparum oocyst loads were then compared between the test and control cohorts. Each replicate comprised of a control cohort injected with GFP dsRNA and the test RNAi cohort – which included the gene of interest; prefoldin subunit-6 and the gene of interest; or prefoldin subunit-6 alone respectively. A Mann- Whitney unpaired t-test was then used to determine significance by calculating the p-value when each of the test cohorts was compared with the control cohort respectively.

The silencing of TEP15 did not show any significant effect (p-value = 0.7345) on mosquito midgut susceptibility to P. falciparum when compared to the control cohort.

Likewise, co-silencing of TEP15 and prefoldin subunit-6 using dsRNA-mediated RNAi did not show any significant deviations in oocyst loads (p-value = 0.3054) when compared with the control group. However, concordant with previous observations (51), silencing of prefoldin subunit-6 (p-value=0.0306) showed a significantly lower oocyst

38 load compared to the control cohort (Figure 3.1).

Silencing of LRIM26 had no significant effect on midgut oocyst load (p-value=

0.4032). Co-silencing of LRIM26 and prefoldin subunit-6 did not have a significant impact on the P. falciparum oocyst load in the midgut of An. gambiae when compared to the control cohort respectively (p-value= 0.8259) (Figure 3.2).

Silencing of enolase (p-value< 0.0001) and the co-silencing of enolase together with prefoldin subunit-6 (p-value= 0.0081) resulted in a significantly lower P. falciparum oocyst load in the midgut of An. gambiae compared to a control cohort silenced with GFP dsRNA. Silencing of enolase showed similar agonistic activity as that identified post- silencing of prefoldin subunit-6 alone (Figure 3.3).

Figure 3.1. Silencing assay of TEP15 gene

RNAi based functional analysis revealed that TEP15 gene is not implicated in anti-Plasmodium defense, nor does co-silencing of TEP15 with PFDN6 affect the latter’s function. The silencing of the PFDN6 as previously shown has an impact on the P. falciparum oocyst loads in mosquito midguts 7-8 days post ingestion of P. falciparum gametocyte-infected blood (*p<.05 in Mann-Whitney unpaired t-test). The points in the graph indicate the absolute oocyst counts in individual mosquitoes treated with the dsRNA indicated on the X-axis. Horizontal red bars in each column represent the median number of oocysts observed in each treatment group. P-values appear below each treatment and refer to that treatment compared to the GFP control.

Figure 3.2. Silencing assay of LRIM26 gene

RNAi based functional analysis indicates that the LRIM26 gene does not play a significant role in the anti-Plasmodium defense of An. gambiae. The silencing of the

PFDN6 as previously shown has an impact on the P. falciparum oocyst loads in mosquito midguts 7-8 days post ingestion of P. falciparum gametocyte-infected blood (*p<.05 in

Mann-Whitney unpaired t-test). The points in the graph indicate the absolute oocyst counts in individual mosquitoes treated with the dsRNA indicated on the X-axis.

Horizontal red bars in each column represent the median number of oocysts observed in each treatment group. P-values appear below each treatment and refer to that treatment compared to the GFP control.

Figure 3.3. Silencing assay of enolase gene

RNAi based functional analysis indicates that the enolase encoding gene plays a significant role in the anti-Plasmodium defense of An. gambiae. Co-silencing of PFDN6 with enolase somewhat rescued the enolase silencing phenotype, probably because of less efficient silencing when dsRNA for two genes are injected simultaneously. The lack of synergistic effect on infection, may suggest that PFDN6 and enolase function in the same mechanism that regulate Plasmodium infection (**p<.01, ***p<0.001 in Mann-Whitney unpaired t-test). The points in the graph indicate the absolute oocyst counts in individual mosquitoes treated with the dsRNA indicated on the X-axis. Horizontal red bars in each column represent the median number of oocysts observed in each treatment group. P- values appear below each treatment and refer to that treatment compared to the GFP control.

Expression of Recombinant Prefoldin Protein Subunit-1 and Subunit-5

The amplified prefoldin subunit gene products and the pET28 vector were subjected to restriction digestion, and the digested products verified visually using agarose gel electrophoresis. Competent bacteria were transformed with the ligated insert- vector plasmid and subsequently examined to confirm transformation. The plasmid extracted from positively transformed colonies was digested with the restriction endonucleases BamHI and Not-I and electrophoresed (Figure 3.4). Successfully transformed colonies were cultured for protein expression. Eluates obtained from the broth cultures were examined using SDS-PAGE to confirm presence of the recombinant protein subunits. The recombinant protein subunit-5, with a molecular weight of

19.05kDa, was successfully eluted with His buffer and stained with coomassie brilliant blue (Figure 3.5). The elution of prefoldin subunit-1 (molecular weight of 14.9 kDa), although expression was observed in the cell pellet (data not shown), was unsuccessful.

Figure 3.4. Analysis of plasmid from transformed colonies

The plasmid was extracted from the transformed colonies and digested with restriction endonucleases BamHI (HF) and NotI respectively and then subjected to agarose gel electrophoresis. The ethidium bromide present in the agarose gel binds to and enables visualization of the nucleic acids. The larger band is the pET28 vector while the smaller band represents the prefoldin subunit-1 (left) gene and prefoldin subunit-5 (right) gene inserts respectively. The prefoldin subunit-1 gene insert size was 387bp while the prefoldin subunit-5 gene insert was 495bp.

Prefoldin Subunit-5 Protein Eluates

Figure 3.5. The above image shows the Tris-Gly gel post electrophoresis comprising of the His-tagged prefoldin subunit-5 (19.9kDa Molecular weight) eluates stained and visualized with coomassie brilliant blue dye. The recombinant prefoldin subunit-5 was obtained from the cell pellet lysate, washed with STE buffer and eluted with the help of

His buffer. The tabular column abbreviation ‘E’ refers to the eluates.

Effect of introduction of recombinant prefoldin subunit-5 on Plasmodium infection

Test cohorts of female An. gambiae were injected with recombinant prefoldin subunit-5 in phosphate buffer saline at concentrations of 0.5mg/mL and 0.25mg/mL. The control cohorts were injected with bovine serum albumin in phosphate buffer saline at concentrations of 0.5mg/mL and 0.25mg/mL respectively. There was no significant difference in the midgut P. falciparum oocyst load between the test cohorts and the control cohorts (Figure 3.6).

A separate experiment was performed, in which the test cohorts were cofed P. falciparum gametocytes with recombinant prefoldin subunit-5 in phosphate buffer saline at concentrations of 0.5mg/mL and 0.25mg/mL and the control cohorts were cofed the gametocytes with bovine serum albumin in phosphate buffer saline at concentrations of

0.5mg/mL and 0.25mg/mL respectively. The first trial showed a significant difference in the midgut oocyst load between the test cohort that was fed with 0.25 mg/mL prefoldin subunit-5 compared to the respective control (Figure 3.7). The second trial for the same concentration indicated that there was no significant difference in the midgut oocyst load of the test and control cohorts (Figure 3.8). In the case of the An. gambiae control cohort fed 0.5mg/mL of prefoldin subunit-5, the Plasmodium infection assay showed no significant differences when compared to the respective control cohort in both trials

(Figure 3.9).

Figure 3.6. Plasmodium infection assay post injection of recombinant prefoldin subunit-5

The control cohorts were injected with bovine serum albumin at concentrations of

0.25mg/mL and 0.5mg/mL respectively. The injection of the recombinant protein at

0.25mg/mL (p-value=0.2833) and 0.5mg/mL (p-value=0.7133) concentration into the thorax of cold anaesthetized female Anopheles does not seem to have a significant impact on the Plasmodium oocyst load in the midgut compared to their respective controls. The points in the graph indicate the absolute oocyst counts in individual mosquitoes injected with the protein indicated on the X-axis. Horizontal red bars in each column represent the median number of oocysts observed in each treatment group. P-values appear below each treatment and refer to that treatment compared to the BSA control.

Figure 3.7. Trial-1: Midgut oocyst loads in Plasmodium infection assay post oral ingestion of recombinant prefoldin subunit-5 (0.25mg/mL concentration)

In the first trial, the P. falciparum infection assay suggested that there is a significant change (p-value= 0.0043) in the median Plasmodium oocyst load in the midgut of the cohort that is co-fed 0.25mg/mL recombinant prefoldin subunit-5 with P. falciparum gametocyte containing blood-serum combination compared to the control cohort which was fed 0.25mg/mL bovine serum albumin with P. falciparum gametocyte containing blood-serum combination. (**p<.05 in Mann-Whitney unpaired t-test). The points in the graph indicate the absolute oocyst counts in individual mosquitoes. The protein and the concentration of the protein that has been fed are indicated on the X-axis.

Figure 3.8. Trial-2: Midgut oocyst loads in Plasmodium infection assay post oral ingestion of recombinant prefoldin subunit-5 (0.25mg/mL concentration)

The second trial indicated there is no significant difference in the midgut oocyst load between control and test cohorts which were co-fed bovine serum albumin

(0.25mg/mL) and recombinant prefoldin subunit-5 (0.25mg/mL) with the parasite gametocytes respectively (p-value=0.1610). The points in the graph indicate the absolute oocyst counts in individual mosquitoes. The protein and the concentration of the protein that has been fed are indicated on the X-axis. Horizontal red bars in each column represent the median number of oocysts observed in each treatment group. P-values appear below each treatment and refer to that treatment compared to the BSA control.

Figure 3.9. Midgut oocyst loads in Plasmodium infection assay post oral ingestion of recombinant prefoldin subunit-5 (0.5mg/mL concentration)

The P. falciparum infection assay suggests that there is no significant impact (p- value= 0.0951) on Plasmodium oocyst load in the midgut of the cohort that is co-fed recombinant prefoldin subunit-5 (0.5mg/mL) with P. falciparum gametocytes compared to the control cohort which was co-fed Bovine serum albumin with the parasite

(0.5mg/mL). The figure and table show pooled data from two trials performed separately.

The points in the graph indicate the absolute oocyst counts in individual mosquitoes. The protein and the concentration of the protein that has been fed are indicated on the X-axis.

DISCUSSION

The identification of LRRD7 as an important immune factor against infection of

Anopheles with Plasmodium, and the subsequent characterization of prefoldin subunit-6 for its interaction with LRRD7, using the yeast two-hybrid system, and later for its agonist-like activity, led to the need for investigating the potential activity of prefoldin and its subunits in Plasmodium infection of the vector Anopheles. Selected proteins from the interactome of prefoldin subunit-6 were further inspected for their role in infection

(52).

The prefoldin subunits 1-6 have been shown to exhibit varying levels of agonist- activity for Plasmodium infection. Prefoldin subunit-5 also exhibited significant agonist- activity, similar to what was seen in experiments with prefoldin subunit-6 using RNAi silencing assays (52). The silencing of prefoldin subunit-5 lowers the Plasmodium oocyst loads of the midgut. The introduction of recombinant prefoldin subunit-5 at a concentration of 0.25mg/mL had conflicting results in the two trials conducted: in the first trial the oocyst load in the midgut of the test cohort were lowered compared to the controls that were fed bovine serum albumin at the concentration of 0.25mg/mL and in the second trial there was no significant difference between midgut oocyst loads. The higher concentration of prefoldin subunit-5 at 0.5mg/mL did not seem to exhibit a similar

59 trend compared to the control bovine serum albumin (0.5mg/mL) fed mosquitoes; in both trials, there was no significant difference in midgut oocyst loads between test and control cohorts. We hypothesize that, the production procedure and resuspension of prefoldin subunit-5 in phosphate buffer saline solution may have resulted in misfolding and abolition of its activity that renders it a P. falciparum agonist. Furthermore, introduction of the recombinant protein through the blood-meal may have resulted in its proteolytic degradation in the midgut lumen. Perhaps, with a different buffer, the solubility and folding of the protein might result in native activity and yield an infection phenotype that agrees with the RNAi-based silencing studies. The high mortality post injection of the recombinant as well as control protein–injected mosquitoes also led to a lesser statistical power in terms of numbers in the injected mosquitoes compared to the ones that co-fed the proteins and the parasite gametocytes. Alternatively, the injection of recombinant protein may not have had any significant impact on P. falciparum infection due to different subcellular localizations compared to the endogenous prefoldin subunit-5.

Although the elution and purification of the prefoldin subunit-5 as well as its effect on the Plasmodium infection of Anopheles was determined, the elution and purification of prefoldin subunit-1 turned out to be difficult. The presence of the recombinant protein’s gene sequence was confirmed in the pET28 vector and the subsequent colony PCR results were positive for the presence of the insert. The presence

60 of the protein was also confirmed in the cell pellet fraction post gel electrophoresis.

However, the conventional techniques and buffers used were ineffective for eluting the recombinant protein from the transformed colonies.

The immune genes identified as part of the interactome of prefoldin subunit-6 were TEP15, LRIM26 and enolase. These genes were silenced by injecting the respective double stranded RNAs into the thorax of cold anaesthetized mosquitoes and compared to a control cohort injected with double stranded RNA of the green fluorescent protein. The silencing of TEP15 did not alter the infection of Plasmodium in Anopheles in contrast to

TEP1, which is a potent anti-Plasmodium immune factor (37). Although TEP family members share high homology, it appears that each of these factors have unique roles in the mosquito. While TEP3 has been shown to have protective activity against the rodent malarial parasite P. berghei, TEP4 has been shown to be protective against the human malarial parasite P. falciparum (51). Concordant with published data, TEP15 does not seem to play a role in the infection of A. gambiae by P. falciparum. The TEP15 gene, present on the right arm of chromosome 3, unlike TEP3 and TEP4 which are found on the left arm, has also been shown to have no vital role in protection against infection by rodent malaria P. yoelii (51).

The silencing of the immune factor LRIM26, discovered for its interaction with prefoldin subunit-6, did not lead to any significant change in the oocyst load of the

61 midgut of infected female mosquitoes compared to the control cohort. The most well characterized members of the leucine-rich repeat immune gene family are APLC1 and

LRIM1 (64). LRIM1 has been categorized as a complement like factor and is known for its involvement in the melanization and lysis of the malarial parasite, in association with

APLC1 and TEP1 (63). The comparison of shared features between LRIM1 and APLC1 led to identification of more than 20 other members of the leucine-rich repeat immune family genes in An. gambiae, Ae. aegypti, C. quinquefasciatus (64). The family members vary in the number of leucine-rich repeats found in them- the “long” LRIMs have 10 or more repeats, the “short” LRIMs have about 6-7 repeats. The up-regulation of several members of the LRIM family including LRIM26 was initiated upon infection by P. berghei in An. gambiae (64). This information coupled with data from the current study implies that while LRIM26 does not play a significant role in the infection of An. gambiae by P. falciparum, but it could be important in infection of the female Anopheles vector by other Plasmodium species.

An. gambiae enolase, the key glycolytic enzyme that mediates conversion of 2- phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), is found ubiquitously in organisms from various phyla (53, 54). The degree of conservation varies between species, and insect enolases show species-specific functions and are less conserved compared to mammalian enolases (54, 55). The invasion of the midgut of female Ae. aegypti by dengue

62 virus or chikungunya virus leads to an increased expression of enolase in the midgut brush border, where it is hypothesized to act as a receptor for the virus (54-57, 62). The enzyme, characterized by its multifaceted roles, is also known to be involved in regulating the transcription of Sendai viral genome. The upregulation of enolase, along with other glycolytic enzymes, during viral invasion of mosquito midgut suggests the possibility of extensive glucose utilization (54). While enolase is primarily involved in energy production, the enzyme has also been found to be present on the surface of

Plasmodium ookinetes (58,59). Previously published data showed that the parasite enolase expressed on the surface of P. berghei is crucial for invasion of oocysts in the midgut of the infected females by binding to the midgut receptor enolase-binding receptor (59). This, however, was not true for P. falciparum oocyst invasion of the midgut, highlighting the possibility that oocyst invasion did not occur via the interaction between the parasite enolase and the midgut receptor (59). The silencing of enolase in Anopheles resulted in decreased oocyst loads in the mosquito midgut, thereby suggesting that mosquito enolase is a P. falciparum agonist. The fact that co-silencing of enolase and PFDN6 did not lead to a stronger (synergistic) suppression of parasites compared to that when the genes are silenced independently may suggest that they are part of the same mechanism, or pathway that regulates Plasmodium infection. PFDN6 silencing has been shown to result in a decreased deposition of laminin on the oocyst surface (Dimopoulos lab,

63 unpublished). Published research has also shown that laminin, a component of the basal lamina, is upregulated after ingestion of a blood-meal (60,61). Similar to PFDN6 and enolase, the silencing of laminin in the mosquito midgut has been shown to decrease the oocyst load (60). One plausible hypothesis is that laminin is permeable to the oocyst capsule since the protein has been spotted on the surface of the oocyst capsule as well as inside the capsule (61). Perhaps laminin and several other host factors such as PFDN6 and enolase play a role in coating and thereby disguising the parasite’s oocyst. The parasite utilizes the components of basal lamina and binds to them, to possibly evade the immune system of the mosquito (60, 61). Further studies are essential to determine if the parasite utilizes mosquito PFDN6 and enolase in a mechanism together with laminin to evade the immune system.

FUTURE DIRECTIONS

The current study and its results highlight the potential of a certain genes as P. falciparum agonist in the African vector An. gambiae. However, the mechanism by which the genes affect parasite infection of the mosquito midgut is still unclear.

My results suggest that enolase may act in the same agonistic mechanism for

Plasmodium as prefoldin. The mechanism can be further elucidated by performing assays to determine the presence of the enolase in the mosquito midgut and its association with the parasite and prefoldin in the midgut. While the results indicate that the other studied immune factors, TEP15 and LRIM26, have no role in the invasion of P. falciparum oocyst in An. gambiae, the possible implication of these genes in the infection of

Anopheles by other Plasmodium species remains unknown.

The exact role of exposure of mosquitoes to the recombinant prefoldin subunit-5 in the Plasmodium infection of Anopheles is unclear, and further refinement of the method and replicates are required to draw conclusions.

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CURRICULUM VITAE

Purnima Ravisankar

Email: [email protected]

Born: 8th March 1994, Chennai, India

Education

Johns Hopkins Bloomberg School of Public Health / Master of Science

August 2015 - May 2017, Baltimore

Rajalakshmi Engineering College / Bachelor of Technology

August 2011 - May 2015, Chennai, India

Research Experience

Johns Hopkins School of Public Health / Master’s Dissertation

November 2015 - PRESENT, Baltimore

Research Advisor: Dr. George Dimopoulos

The role of immune genes in the transmission of the malarial parasite Plasmodium falciparum by Anopheles gambiae are being studied using RNAi. Simultaneously, recombinant mosquito prefoldin is being expressed in transformed bacteria and its effect on pathogen transmission will be studied. 76

Research Experience

Wellcome Trust Research Laboratory / Summer Research Fellowship

June 2014 -August 2014, Vellore, India

Research Advisor: Dr. Gagandeep Kang

The serotypes of the Rotavirus were identified using a combination of Genetic

Engineering and Molecular Biology.

Sankara Nethralaya / Undergraduate Thesis

January 2015 - April 2015, Chennai, India

Research Advisor: Dr. R. Malathi

Clinical isolates were tested phenotypically and genotypically to detect the production of β-lactamase enzymes which confer resistance toward β-lactam antibiotics.

Certifications

Protein Purification Techniques

Aristogene Biosciences, Bangalore

December 2012

Awards

● Recipient of the Summer Research Fellowship in the year 2014 at Christian

Medical College, Vellore

● Recipient of the ScM 2nd year tuition fellowship at JHSPH

● Anna University Rank Holder in B.Tech Biotechnology batch of 2011-2015

● Secured the 3rd place in Bio-Business Challenge at the 2014 inter-collegiate

symposium held at St. Joseph’s College of Engineering

● Awarded the “Raghavan Memorial Merit Award” for being the overall topper

in Rajalakshmi Engineering College amongst freshman.