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No description available.

Malaria is caused by a protozoan parasite belonging to the genus Plasmodia. Plasmodium falciparum is responsible for the fatal form of human malaria. Spread of drug resistant parasites warrants for sound biological understanding of the parasite at both cellular and biochemical level. Heat shock proteins are highly conserved group of proteins required for correct folding, transport, and degradation of substrate proteins in vivo. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where in cooperation with Hsp10, it participates in protein folding. Keeping in mind the central importance of chaperones in biological processes, our lab has been interested in examining roles of heat shock proteins in malarial parasite during its asexual growth in human erythrocytes. During its life cycle, the parasite continually shuttles between a cold-blooded insect vector with the body temperature of 27°C and a warm-blooded human host with the body temperature of 37°C and parasite experiences episodes of heat shock periodically. Therefore malaria parasite serves as good model to study heat shock protein functions. Like all biological systems, the malaria parasite expresses several chaperones including proteins of the Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100 families. Towards this we have systematically characterized different families of stress proteins Hsp40, Hsp60, Hsp70, Hsp90 as well as Hsp100. In addition to cloning their genes we have studied their expression, localization, abundance, complexes and their biological roles. Earlier studies from our lab showed PfHsp90 is essential for parasite growth and survival in human erythrocytes. Our present study attempts to study heat shock protein 60 of the malarial parasite (PfHsp60). In this connection we have been successful to clone and express PfHsp60 gene from Plasmodium falciparum in E. coli and to raise antibodies specific to PfHsp60. We have examined its expression and import in the mitochondrion of malarial parasite during its asexual growth in human erythrocytes. Analysis of the total parasite lysates resolved by two-dimensional gel electrophoresis followed by western blotting using specific antibodies showed PfHsp60 exhibits an isoelectric point corresponding to its signal uncleaved precursor (pI - 6.2). Mass spectrometric analysis of the spot corresponding to precursor PfHsp60 confirmed the presence of signal peptide region. Co-immunoprecipitation analysis of total parasite lysates with antibodies specific to PfHsp60 showed precursor PfHsp60 to be associated with PfHsp70 and PfHsp90. Co-immunoprecipitation from the mitochondrial and cytoplasmic fraction confirmed the position of mature PfHsp60. Indirect immunofluorescence analysis also showed presence of a pool of PfHsp60 in the cytoplasm of the parasite, in addition to its expected localization in the mitochondrion. Treatment of parasite infected erythrocytes with an inhibitor of Hsp90 disrupted its association with cytoplasmic chaperones and targeted precursor Pfhsp60 for intracellular degradation. On the other hand treatment with the mitochondrial import inhibitor further inhibited the import of precursor PfHsp60 into the mitochondrion and stabilized its interaction with cytosolic chaperones. Previous reports have shown that there are four fold accumulations of PfHsp60 transcripts in heat shocked parasites. However, the expression of PfHsp60 was not induced upon heat shock in the blood stages of P.falciparum. Biochemical data indicate that the mitochondrion is not the source of ATP in the parasite. Furthermore the genome does not seem to encode the critical subunits of Fo-F1 ATP synthase. Yet, the active mitochondrial electron transport chain serves for regeneration of ubiquinone required for pyrimidine biosynthesis. The active electron transport chain is critical for parasite survival. Recent study with the lab-grown 3D7 strain of malaria parasite concluded that mitochondria are not required for energy conversion. Transcriptome analysis of the parasite derived directly from blood samples of infected patients showed that genes encoding the proteins of mitochondrial biogenesis, oxidative phosphorylation, respiration and highlighted the mean expression level for PfHsp60 is dramatically up regulated in parasites. Gene up regulation doesn’t always translate to increase in protein function or metabolic up regulation. When we analyzed the total parasite lysates of field isolates resolved by two-dimensional gel electrophoresis also showed presence of the precursor form of Pfhsp60 in the cytoplasm of the parasite. Overall, our observations indicated accumulation of precursor PfHsp60 in the parasite cytoplasm suggesting an inefficient mitochondrial protein import in the malarial parasite. The defect in mitochondrial protein import is possibly reflective of the compromised energy state of the parasite mitochondrion. This fits with the model that has been reported in mutant strains of yeast, Saccharomyces cerevisiae lacking functional F o-F1-ATPase. These strains were found to grow very poorly under anaerobic conditions and are known to accumulate Hsp60 protein in the cytoplasm mainly its precursor form. Under optimal growth conditions most eukaryotes maintain close co-ordination between gene expression, translation and translocation efficiently. As a result, mitochondrial precursor proteins are usually not found to accumulate in the cytoplasm. To our knowledge this the first report suggesting an inefficient co-ordination in the synthesis and translocation of precursor PfHsp60 and possibly other proteins during asexual growth of malarial parasite in human erythrocytes under optimal growth conditions. Finally, expression of the PfHsp60 gene in E.coli resulted in its association with bacterial GroEL subunits co-fractionating with a size of 920 kDa, corresponding to the tetra decameric form. The observation indicated possible existence of a hybrid chaperonin complex consisting of subunits from ectopically expressed PfHsp60 and endogenous GroEL.

Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.

Molecular chaperones are a group of proteins which maintain cellular homeostasis by assisting de novo protein folding and their refolding to native state after destabilization due to external stress. They are also known as heat shock proteins as they were first discovered as a response to heat stress. It is now well established that the function of this group of proteins is not only restricted to protein homeostasis but also extends to diverse cellular processes such signal transduction, development and differentiation. Heat shock protein 90 (Hsp90) is one of the most abundant molecular chaperones that is highly conserved from prokaryotes to eukaryotes. Hsp90 is an essential chaperone and is required for the viability of all eukaryotes examined so far including yeast, Drosophila and Caenorhabditis elegans. Hsp90 has emerged as an important regulator of cellular activities by virtue of its ability to interact with a diverse set of client proteins many of which include transcription factors, protein kinases and signaling molecules. Through interaction with these proteins it is involved in regulating cellular processes including growth, cell cycle, endocrine functions, apoptosis, differentiation and development. Further in Drosophila and plants, Hsp90 is thought to function as a capacitor for morphological evolution and phenotypic variation. Recently, it has also been implicated in the emergence of drug resistance in Candida albicans. Furthermore, the importance of Hsp90 in disease states, particularly in cancer, is strongly evident, where chaperoning of mutated and oncogenic proteins is critical for continuous proliferation of cells. This has led to the development of Hsp90 inhibitors as an anti-cancer drug. Geldanamycin (GA), a benzoquinone ansamycin was the first molecule shown to inhibit Hsp90 activity by binding to its ATP binding domain. A derivative of GA, 17-allylamino-17-demethoxygeldanamycin (17AAG), has shown promise in clinical studies and has entered Phase III clinical trials. Hsp90 has been shown to be important for growth and development of many protozoan parasites. Inhibition of Hsp90 function in Leishmania, Emiera, Toxoplasma, Trypanosoma as well as Plasmodium causes a block in their developmental cycle. Previous studies from our laboratory have shown that inhibition of Hsp90 function prevents growth of malaria parasite in human erythrocytes in vitro. P. falciparum Hsp90 (PfHsp90) has also been shown to regulate parasite growth during the febrile episodes that are characteristic of malaria. While most of the studies highlighting the importance of PfHsp90 have relied on its pharmacological inhibition, its biochemical characterization and quantitative measurement of its interaction with GA in isolated system has not been explored. It was also not understood whether the in vitro model of Hsp90 inhibition could translate into inhibition of the parasite growth in an animal model of malaria. Since Hsp90 is a split ATPase requiring proper co-ordination between the residues on its N-terminal and middle domains, it would be desirable to biochemically characterize full length PfHsp90 to gain insights into its potential as an anti-malarial target. The present study was initiated with an objective of understanding the biochemical properties of Hsp90 from P. falciparum in terms of ATP binding, ATP hydrolysis and its GA binding ability. We have also examined the potential of PfHsp90 to serve as a chemotherapeutic target using its clinically well-established inhibitor, 17AAG, in a preclinical mice model. Apart from using in vitro and in vivo models of malaria, we have also explored the efficacy of 17AAG in the P. falciparum samples collected from malaria patients. Additionally, we have examined the relevance of chaperones, in particular PfHsp90 in the samples collected from malaria patients. Finally, we have attempted to understand the unexplored biology of another malaria parasite P. vivax by a high throughput proteomics approach. Biochemical characterization of PfHsp90 and its comparison with host Hsp90 Hsp90 belongs to GHKL (gyrase, Hsp90, histidine kinase, MutL) protein family having a characteristic novel ATP-binding Bergerat fold. The ATP binding pocket of GHKL family differs from the conventional nucleotide binding fold in the formation of a cone shaped pocket made up of four anti-parallel β-sheets and three α helices as opposed to parallel βsheets surrounded by α-helices in the latter. The most distinctive feature of Bergerat fold is the presence of ATP lid. Further, even within the GHKL family members the composition and the conformation of this ATP-lid differs, leading to different solvent exposure of the bound ATP. All Hsp90s from different organisms, characterized so far, have been shown to posses ATP binding and hydrolysis activity but so far PfHsp90 ATPase activity has not been characterized. Using intrinsic tryptophan fluorescence measurements, we found PfHsp90 to bind ATP with about 30% higher affinity than human Hsp90 (hHsp90). We further, 32 determined the ATPase activity of PfHsp90 by monitoring the direct conversion of (γ-P) 32-2 ATP to Pi. PfHsp90 bound and hydrolyzed ATP with a Km of 611 µM and kcat of 9.9 x 10 -1m . Interestingly, PfHsp90 showed six times higher ATPase activity as compared to its human homologue and more intriguingly the ATPase activity exhibited by PfHsp90 was highest among all the Hsp90s studied so far. Previous studies from our laboratory have provided sufficient evidence for inhibitory action of GA on Plasmodium growth inside the infected erythrocytes. GA is known to exert its inhibitory effect by binding to the ATP binding domain of Hsp90 thus inhibiting its chaperone activity. Earlier reports have shown that despite a high similarity between the ATP/GA binding region in Hsp90 from different organisms, there is a difference in their ability to bind GA. For example, in spite of all the hallmarks of ATP-binding pocket of Hsp90 family C. elegans Hsp90 does not bind GA. We have employed fluorescence spectroscopy to examine whether PfHsp90 can bind to GA. In parallel, we have also determined the binding affinity of human Hsp90 (hHsp90) to GA. We observed small but reproducible differences in the binding affinity of GA to Hsp90s from human host and P. falciparum with latter having fourfold higher affinity. A sequence analysis of the GA binding domain of Hsp90s from P. falciparum and human host showed a homologous substitution of K112 of hHsp90 to R98 in PfHsp90. In order to examine the effect of this substitution, if any, on the observed difference in GA binding abilities, we mutated R98 to K in PfHsp90. However, we did not find any difference in the binding ability of R98K PfHsp90 to GA, suggesting that this homologous substitution has minimal or no effect on drug protein interaction in vitro. However, in view of this phylogenetically conserved substitution, we cannot rule out its role in vivo. The chaperone function of Hsp90 is dependent on its ATPase activity which is susceptible to GA mediated inhibition. We next examined the extent of inhibition of GA on the ATPase activity of Hsp90s from P. falciparum and human host. Interestingly, we found the PfHsp90-ATPase activity to be three times more sensitive than hHsp90-ATPase activity to GA mediated inhibition suggesting that the malaria parasite, P. falciparum is likely to be more sensitive to GA when compared to human host. This result is in accordance with a recent study, which has shown that yeast expressing PfHsp90 in lieu of native yeast Hsp90 was more sensitive to GA than yeast expressing either yeast Hsp90 or human Hsp90. Acetylation of Plasmodium falciparum Hsp90 Post-translational modification of Hsp90 such as acetylation has been shown to affect its binding with GA. We first examined whether, PfHsp90 can be acetylated. With the use of various purified Histone acetyl transferases (HATs) of human origin, we have shown PfHsp90 to undergo acetylation in vitro. We found that among different HATs (pCAF, Gcn5 and p300) used, only p300 was able to acetylate PfHsp90 suggesting a role for it in PfHsp90 in vivo acetylation as well. We next examined the in vivo acetylation status of PfHsp90 from parasite lysate. To enrich the acetylated fraction of PfHsp90, we have used Histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Immunoprecipitation of PfHsp90 followed by immunoblotting with an acetyl-lysine antibody confirmed that PfHsp90 undergoes acetylation in vivo. In order to identify the lysine residues which underwent acetylation we subjected the acetylation enriched fraction of PfHsp90 to in-gel trypsin digestion followed by mass spectrometry. Analysis of trypsin digested PfHsp90 from the parasites identified three sites of acetylation, one of which overlapped with PfHsp90 cochaperone (Aha1 and p23) binding residue, suggesting that acetylation could play a potential role in modulating PfHsp90 multi-chaperone complex assembly. Indeed, treatment of P. falciparum cultures with a HDAC-inhibitor resulted in partial dissociation of PfHsp90 complex as observed from size-exclusion chromatography. Adding to this observation, we also found that co-treatment of TSA and GA showed a synergistic and additive effect in inhibiting parasite growth in vitro. The above results suggest the possibility of using Hsp90 inhibitor in combination with HDAC inhibitor to arrest Plasmodium growth and development. Clinically tested GA-analogue 17AAG inhibits Plasmodium growth in vitro and in vivo The specificity of GA inside the cell has been a matter of debate since the discovery of its medicinal importance. In the past, Hsp90 has been implicated as a target of GA by carrying out immunoblotting of GA pull-down fraction with an anti-Hsp90 antibody. Crystal structure of GA with yeast Hsp90 has shown it to bind within the well conserved ATP-binding pocket of Hsp90. However, the specificity of GA inside the cell is still a conjecture. We have performed GA pull down assays from the parasite lysate followed by Coomassie Blue staining, which gave a single band corresponding to 86 kDa PfHsp90. The identity of PfHsp90 was further confirmed by immunoblotting with antibody specific to PfHsp90. This result indicates that inside the cells, inhibitory effect of GA is mediated by and large through its interaction with Hsp90. However, we cannot rule out the presence of other minor, less significant, interactors of GA. Earlier work from our laboratory has shown that GA inhibits Plasmodium growth inside the infected erythrocytes. However, issues related to GA toxicity have excluded its development as a therapeutic. Nevertheless, interest in this class of molecule has led to the generation of a large number of less toxic derivatives of GA. One classical example is 17AAG which has gained clinical importance over the years and has entered in phase III trial. Intrigued by the clinical success of 17AAG, we were interested in determining its ability to modulate parasite growth. Indeed, 17AAG was able to inhibit parasite growth in a manner similar to that of GA. We further extended our study to parasites isolated from patient samples. Here too, we found 17AAG to be effective in inhibiting growth of the parasite. Finally, we examined the efficacy of 17AAG at a pre-clinical level using a mouse model of malaria. Using Peters’ four-day test we found 17AAG, to be effective in attenuating parasite growth and prolonging the survival of parasite infected mice (n=4, p=0.00692; n=10, p=0.001). Clinical relevance of heat shock proteins of Plasmodium falciparum A recent study using in vivo expression profiles of parasites derived from blood samples of infected patients has revealed previously unknown physiological diversity in the biology of malaria parasites. According to gene expression profiles, parasites were clustered into three different physiological states – starvation, glycolysis dependent active growth and environmental stress response. In order to examine the clinical relevance of molecular chaperones in malaria, we reanalyzed the previously published gene expression data of clinical parasites from 46 patients. Our analysis of this data showed that organellar chaperones were up-regulated upon starvation (cluster1) while cytosolic chaperones such as Hsp90 were up-regulated in active growth conditions (cluster2) indicating up-regulation of distinct group of Hsps in response to different environmental cues. Interestingly, Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, clustered in the same group. Further, some patients of cluster 3 (environmental stress response) showed higher expression of Hsp90 while others showed lower expression. In general, cluster 3 group of patients were heterogeneous in terms of expression of chaperones. Using non-negative matrix factorization (NMF), cluster 3 was sub-clustered into two groups 3a and 3b. Cluster 3b showed up-regulation of cytosolic chaperones similar to cluster 2 indicating these two clusters are inter-related. Most of the Hsp90 dependent pathways such as trafficking, signaling, anti apoptotic and pro-survival found to be most active in cluster 2 indicating the dependence of this group of parasites on Hsp90. The two main outcomes of our chaperone analysis are (1) the up-regulation of molecular chaperones in parasites are not a general response to hostile conditions as perceived previously, but is largely determined by the host factors and may differ from one host to another (2) the disease specific pathways may exist in natural condition by the up-regulation of specific chaperone and its interactors as a response to different host environment. Clinical proteomics of human malarial parasites Much of our understanding about the life cycle of parasites and importance of parasite proteins have been gleaned from the studies in laboratory strain or with the laboratory adapted clinical parasites. Although, these studies provide us first hand information about the functionality and the importance of these proteins, but they often fail to mimic the actual disease environment. In the patient, parasites are exposed to host factors such as hormones, metabolites, inflammatory mediators which can influence the expression of proteins and thus parasite biology. Further, instead of parasite exposure to 37°C temperature throughout the erythrocytic cycle in vitro, it is exposed to several rounds of febrile episodes inside human, which can also influence the parasite life cycle. Furthermore, clinical analysis is important to validate the presence and expression of drug targets in actual disease environment. Therefore, analysis of malaria parasite from clinical settings has become an important component in our laboratory and this thesis. Proteomic analysis of clinical samples has emerged as an important tool to understand the proteins dynamicity as response to disease environment. We have initiated clinical proteomic study of P. falciparum, the cause of most common and fatal malaria in humans and extended it further to the neglected malaria parasite P. vivax. The study of P. vivax has largely been over-shadowed by the enormous attention devoted to P. falciparum. Notably, the drugs which have been discovered against P. falciparum are not as effective against P. vivax. Further several unique features of P. vivax such as dormant hyponozoites, reticulocyte host preference and formation of specialized caveolae vesicle complex structure distinguish its biology from P. falciparum and warrant concerted effort directed at this parasite. A major limitation in studying this parasite is the absence of a long-term culturing system. Therefore, research on this parasite requires samples obtained directly from patients. In spite of the inherent difficulty in obtaining such samples, this method provides us an opportunity to study this parasite in its real environment which has a huge effect on the expression as well as function of parasites and host proteins. Our current knowledge about the life cycle of this parasite has been gained from the recently published transcriptome study. Even though transcriptome analyses provide useful understanding at the level of gene expression, they do not reflect the active protein component of a cell. In other words, most of disease outcome is a result of interaction of the protein component with the environment. We therefore attempted to understand the protein component of this parasite in the disease environment to shed light on its pathogenicity. Despite facing several challenges in the way of proteomic analysis of this parasite such as availability of samples, low parasitemia, contamination of parasite proteins with abundant host proteins etc, we were able to identify 154 P. vivax proteins abundantly expressed in clinical environment using mass-spectrometry based approach. We found many proteins unique to this parasite along with known drug targets. This study is the first of its kind and could prove to be a very important step towards gaining insights into the physiology of this parasite.This study serves as a proof-of-principle method which in future is likely to help in identifying many more potential drug targets, vaccine candidates and diagnostic markers from clinically relevant samples as opposed to cultured samples. Summary Despite the importance of PfHsp90 in malaria biology, it has not been characterized in terms of its biochemical properties and its interaction with the inhibitor. In this study, we have successfully cloned, expressed, purified and characterized full length PfHsp90. We found that PfHsp90 exhibits a hyper-ATPase activity and is more sensitive to GA mediated inhibition as compared to human Hsp90. We have also shown that its sensitivity towards GA is dependent on its acetylation status as treatment of infected erythrocytes with HDAC inhibitors increases its sensitivity to GA. Using a pull-down assay, we have determined, unequivocally, that GA specifically binds to Hsp90. Most importantly, we have demonstrated that 17AAG, a clinically well-established inhibitor of Hsp90, inhibits parasite growth in a laboratory strain, field isolates and an in vivo mouse model of malaria. Overall, our biochemical characterization and drug interaction studies underscore the importance of PfHsp90 as a potent drug target and its inhibitors as a candidate drugs for the treatment of malaria, one of the deadly human infectious diseases. Our efforts to understand the importance of molecular chaperones in parasites isolated directly from patient samples (clinical setting) has revealed conspicuous association of Hsps with previously defined parasite physiological states. In particular, parasites obtained from a specific group of patients exhibited heightened dependence on Hsp90-dependent pro-survival pathways, indicating an increased response to host stressors in this group of parasites. Thus, parasite encoded chaperones, in particular PfHsp90, play a major role in defining the pathogenesis of malaria. A disease is an outcome of interaction between pathogens and its host, therefore it is important to study parasite in its real environment to understand disease pathogenesis. Our lab has previously reported the first ever proteomic analysis of P. falciparum from malaria patients. In this study, we have made an attempt to understand the unexplored biology of another important malaria parasite P. vivax. We have used a mass-spectrometry based approach to identify the protein content of this parasite. This technically challenging attempt has enabled us to identify many proteins. This study is an important step towards understanding the biology of this parasite in dearth of any information available on the proteins involved in this parasite’s pathogenicity.

Molecular chaperones have emerged in recent years as major players in many aspects of cell biology. Molecular chaperones are also known as heat shock proteins (HSPs) since many were originally discovered due to their increased synthesis in response to heat shock. They were initially identified when Drosophila salivary gland cells were exposed to a heat shock at 37°C for 30 min and then returned to their normal temperature of 25°C for recovery. A “puffing” of genes was found to have occurred in the chromosome of recovering cells, which was later shown to be accompanied by an increase in the synthesis of proteins with molecular masses of 70 and 26 kDa. These proteins were hence named “heat shock proteins”. The first identification of a function for HSPs was the discovery in Escherichia coli that five proteins synthesized in response to heat shock were involved in λ phage growth. The products of the groEL and groES genes were found to be essential for phage head assembly while the dnaK, dnaJ and grpE gene products were essential for λ phage replication. It was later shown that GroEL and GroES are part of a chaperonin system for protein folding in the prokaryotic cytosol while DnaK is a member of the Hsp70 family that works in conjunction with the DnaJ (Hsp40) co-chaperone and the nucleotide exchange factor GrpE to promote phage replication by dissociating the DnaB helicase from the phage-encoded P protein. Since then, a large number of other proteins collectively referred to as HSPs have been discovered. However, heat shock is not the only signal that induces synthesis of heat shock proteins. Stress of any kind, such as nutrient deprivation, chemical treatment and oxidative stress among others causes increased production of HSPs and therefore, they are also known as stress proteins. The term “molecular chaperone” was originally used to describe the function of nucleoplasmin, a Xenopus oocyte protein that promotes nucleosome assembly by binding tightly to histones and donating the bound histone to chromatin. However, since then, chaperones have been defined as “a family of unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structure”. This view of molecular chaperones, though undoubtedly correct, doesn’t capture the multifaceted roles they have since been discovered to play in cellular processes. In recent years, molecular chaperones have been shown to perform other functions in addition to the maintenance of protein homeostasis: translocation of proteins across organelle membranes, quality control in the endoplasmic reticulum, turnover of misfolded proteins as well as signal transduction. As a result, many chaperones are also essential under non-stress conditions and play crucial roles in cell growth and development, cell-cell communication and regulation of gene expression. Heat shock protein 90 (Hsp90) is one of the most abundant and highly conserved molecular chaperones in organisms ranging from bacteria to all branches of eukarya. It has been shown to be essential for cell viability in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster. Although the bacterial homolog HtpG is dispensable under normal conditions, it is important for cell survival during heat shock. In addition to its role as general chaperone in protein folding following stress, Hsp90 has a more specialized role as a chaperone for several protein kinases and transcription factors. Many Hsp90 client proteins are signaling proteins involved in regulation of cell growth and survival. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance resulting in a key role for Hsp90 in these processes. Recent reports have also highlighted a role for Hsp90 in linking the expression of genetic and epigenetic variation in response to environmental stress with morphological development in Drosophila melanogaster and Arabidopsis thaliana. In Candida albicans, Hsp90 augments the development of drug resistance, implicating a role for Hsp90 in the evolution of infectious diseases. The malarial parasite, Plasmodium falciparum, is the causative agent of the most lethal form of human malaria. The parasite life cycle involves two hosts: an invertebrate mosquito vector and a vertebrate human host. As the parasite moves from the mosquito to the human body, it experiences an increase in temperature resulting in a severe heat shock. The mechanisms by which the parasite adapts to changes in temperature have not been deciphered. Our laboratory has been interested in investigating the role of heat shock proteins during acclimatization of the parasite to such temperature fluctuations. Heat shock proteins of the Hsp40, Hsp60, Hsp70 and Hsp90 families have been characterized in the parasite and are being examined in our laboratory. This thesis pertains to understanding the functional role of Plasmodium falciparum Hsp90 (PfHsp90) during adaptation of the parasite to fluctuations in environmental temperature. The parasite expresses a single gene for cytosolic Hsp90 on chromosome 7 (PlasmoDB accession no.: PF07_0029) coding for a protein of 745 amino acids with a pI of 4.94 and Mw of 86 kDa. Eukaryotic Hsp90 regulates several protein kinases and transcription factors involved in cell growth and differentiation pathways resulting in a crucial role for Hsp90 in developmental processes. A role for PfHsp90 in parasite development, therefore, seems likely. Indeed, PfHsp90 has previously been implicated in parasite development from the ring stage to the trophozoite stage during the intra-erythrocytic cycle. Pharmacological inhibition of PfHsp90 function using geldanamycin (GA), a specific inhibitor of Hsp90 activity, abrogates stage progression. These experiments suggest that PfHsp90 may play a critical role in parasite development. This is further substantiated by the fact that several pathogenic protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Toxoplasma gondii and Eimeria tenella depend on Hsp90 function during different stages of their life cycles. It appears, therefore, that a principal role of Hsp90 in protozoan parasites may be the regulation of their developmental cycles. However, the precise functions of PfHsp90 during the intra-erythrocytic cycle of the malarial parasite are not clear. In this study we have carried out a functional analysis of PfHsp90 in the malarial parasite. We have examined the role of PfHsp90 in parasite development during repeated exposure to febrile temperatures. We have investigated its involvement in parasite development during a commonly used synchronization protocol involving cyclical changes in temperature. We have examined the interaction of GA with the Hsp90 multi-chaperone complex from P. falciparum as well as the human host. Finally, we have carried out a systems level analysis of chaperone networks in the malarial parasite as well as its human host using an in silico approach. We have analyzed the protein-protein interactions of PfHsp90 in the chaperone network and predicted putative cellular processes likely to be regulated by parasite chaperones, particularly PfHsp90.