INTRODUÇÃO: As proteínas de choque térmico (HSPs) são proteínas presentes em todos os seres vivos. Elas possuem dupla atividade imunológica, induzindo tanto respostas proinflamatórias quanto imunorreguladoras. Em nosso grupo, estudamos os efeitos imunológicos dos peptídeos da HSP60 nos sistemas humano e murino. OBJETIVOS: Identificar peptídeos da HSP60 potencialmente imunorreguladores no sistema murino, visando a indução de tolerância no contexto do alotransplante. MÉTODOS: Analisamos a capacidade dos diferentes peptídeos de modificar, in vitro a expressão de genes predominantemente imunorreguladores (Foxp3, Gata-3, IDO, IL-4, IL-10, TGF?) ou inflamatórios (T-bet, IL-12p35, IL-12p40, IL-17) por PCR de tempo real, em esplenócitos murinos de animais naive. Consideramos as modificações induzidas pelos peptídeos como REGULA (?expressão de genes imunorreguladores ou ?expressão de genes inflamatórios) ou INFLAMA (?expressão de genes imunorreguladores ou ? expressão de genes inflamatórios). Selecionamos peptídeos que induziram predomínio de modificação da expressão gênica do tipo REGULA, mais promissores como imunorreguladores para ensaios in vivo de indução de tolerância ao aloenxerto de pele murino. RESULTADOS: Os peptídeos mais promissores foram N7, C4, N6, I8, N2 e N3. O peptídeo N7(n=9) induziu uma razão REGULA/INFLAMA=2,71 e induziu a redução da expressão gênica relativa de IL-17 e IL-12p40. O peptídeo C4 (n=6) induziu razão REGULA/INFLAMA=1,63 e induziu redução da expressão gênica relativa de IL-17 e IL-12p40. O peptídeo N6 (n=8) induziu uma razão REGULA/INFLAMA=1,60, induziu a redução da expressão gênica relativa de T-bet e IL-12p40 e aumentou a expressão de IL-10. O peptídeo I8 (n=5) induziu razão REGULA/INFLAMA=1,57 e induziu aumento da expressão gênica relativa de IL-17. O peptídeo N2 (n=8) induziu razão REGULA/INFLAMA=1,32 e induziu redução da expressão gênica relativa de IL-17 e aumento da expressão de IL-10, IL-4 e T-bet. O peptídeo N3 (n=7) induziu razão REGULA/INFLAMA=1,25 e não teve destaques na modificação gênica induzida. O peptídeo N7 foi testado em protocolos para indução de tolerância do alotransplante de pele murino. Não houve diferença estatística entre os diferentes grupos, a despeito do aumento de sobrevida do aloenxerto de 7 dias no protocolo em combinação com o anticorpo ?CD3 (p=0,38, n=5). DISCUSSÃO E CONCLUSÃO: O forte impacto imunorregulador de alguns peptídeos da HSP60 na expressão gênica de moléculas imunológicas indica que eles podem ser explorados em imunoterapias. Apesar da existência de variabilidade individual, o peptídeo N7 da HSP60 parece ser um promissor peptídeo imunorregulador. O discreto aumento da sobrevida do aloenxerto de pele induzida pelo peptídeo N7 sugere que a sua combinação com imunossupressores em outros protocolos de indução de tolerância, pode ser uma estratégia útil para aumentar a sobrevida do aloenxerto / INTRODUCTION: Heat shock proteins (HSPs) are proteins present in all living beings. They have dual immunologic functional activity inducing both proinflammatory and regulatory responses. In our group, we study the immunological effects of HSP60 peptides in human and mice. OBJECTIVES: To identify potentially immunoregulatory HSP60 peptides in mice, aiming at tolerance induction in the context of alotransplantation. METHOD: We analyzed the capacity of different HSP60 peptides to modify in vitro the expression of predominantly immunoregulatory genes (Foxp3, Gata-3, IDO, IL-4, IL-10, TGF?) or inflammatory (T-bet, IL-12p35, IL-12p40, IL-17) by Real Time PCR, in naive mice splenocytes. We considered gene modifications induced by the peptides as REG (?expression of immunoregulatory genes or ?expression of inflammatory genes) or INFLAMMA (?expression of immunoregulatory genes or ? expression of inflammatory genes). We selected some promising peptides that induced predominantly REG gene expression modifications as immunoregulatory peptides, to perform in vivo assays of tolerance induction to murine skin allograft. RESULTS: More promising peptides were N7, C4, N6, I8, N2 and N3. The N7 peptide (n=9) induced a REG/INFLAMMA ratio=2,71 and induced gene expression reduction of IL-17 and IL-12p40. The C4 peptide (n=6) induced a REG/INFLAMMA ratio =1,63 and induced gene expression reduction of IL-17 and IL-12p40. The N6 peptide (n=8) induced a REG/INFLAMMA ratio =1,60, induced gene expression reduction of T-bet and IL-12p40, and increased expression of IL-10. The I8 peptide (n=5) induced a REG/INFLAMMA ratio =1,57 and induced increased gene expression of IL-17. The N2 peptide (n=8) induced a REG/INFLAMMA ratio =1,32 and induced gene expression reduction of IL-17 and increase expression of IL-10, IL-4 and T-bet. The N3 peptide (n=7) induced a REG/INFLAMMA ratio =1,25 and induced no gene expression modification highlights. The N7 peptide was tested in tolerance mice allograft induction protocols. There was no statistical difference between different groups, despite the 7-day increase in allograft survival with the protocol in combination with the ?CD3 antibody (p=0,38, n=5). DISCUSSION AND CONCLUSION: The strong immunoregulatory impact of some HSP60 peptides in the gene expression of immune molecules indicate that they may be explored for immune therapy. Despite the existence of some interindividual variability, the N7 HSP60 peptide seems to be a promising imunorregulatory peptide. The slight increase in graft survival with the N7 peptide suggests that the combination of HSP60 peptides with other immunosupressors and different tolerance induction protocols may be a way to improve graft survival.

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.

Chaperonins (Hsp60 chaperones) comprise a class of oligomeric, high-molecular-weight chaperones that have the unique ability to fold some proteins that cannot be folded by simpler chaperone systems. The term &ldquo / thermosome&rdquo / is used for molecular chaperonins from Archaeal organisms since they accumulate to high levels upon heat-shock. In this study first time, we have cloned and sequenced two Hsp60 subunit genes (&amp / #945 / and &amp / #946 / ) from a thermoacidophilic archaeon Thermoplasma volcanium. For cloning we have followed a PCR based strategy. Amplification of Hsp60 &amp / #945 / gene from chromosomal DNA of T. volcanium yielded a product of 1939 bp amplicon and that of Hsp60 &amp / #946 / gene yielded a product of 1921 bp amplicon. After ligation of the PCR fragments to pDrive vector, recombinant plasmids were transferred into E. coli TG-1 competent cells and recombinant colonies were selected by blue/white screening. The cloning of two subunit genes were confirmed by restriction mapping and by sequencing. Both subunit genes were then subcloned to pUC18 vector consequtively to construct a co-expression vector. Both subunit genes were expressed under control of their own promoters leading to production of active Hsp60 chaperonin (thermosome). Chaperone activity of the recombinant thermosome was shown by using pig citrate synthase enzyme as substrate. Thermosome induced refolding was observed when renaturation was carried out at 50&deg / C for 2,5 h. Under this condition, citrate synthase activities associated with control and test were &amp / #61508 / mA412/min:19.0 and &amp / #61508 / mA412/min:24.0 respectively. Clustal W Version 1.82 was used for multiple sequence alignments of Hsp60 &amp / #61537 / and Hsp60 &amp / #61538 / proteins of T. volcanium and other Hsp60 proteins from various eukaryotes, bacteria and archaea. The highest sequence similarity was found between &amp / #945 / subunit proteins of T. volcanium and T. acidophilum (94%) and &amp / #946 / subunit proteins of T. volcanium and T. acidophilum (93%). Clusters of orthologous groups and conserved domain database searches revealed the phylogenetic relationships between Hsp60 &amp / #61537 / and Hsp60 &amp / #61538 / subunits of T. volcanium thermosome and other Hsp60 proteins from various eukaryotes, bacteria and archaea. Induction of both subunit genes under heat shock (65&deg / C, 70&deg / C and 75&deg / C for 2h) and under oxidative stress (imposed by 0,008 mM, 0,01 mM, 0,02 mM, 0,03 mM and 0,05 mM H2O2) conditions was studied by Real-Time PCR technique and amplified cDNA band density analysis.

A Chlamydia trachomatis heat shock protein 60kDa (chsp60) exhibits increased expression in response to iron limitation. Genome sequencing revealed three genes encoding chsp60s. The objective of this study was to generate peptide antisera that would selectively recognize each chsp60. The DNA sequence for each C. trachomatis serovar E chsp60 was determined and compared with existing genome sequences. Predictive amino acid sequences were evaluated for peptides unique to each chsp60. Synthetic peptides were used to generate antisera; the resultant sera were purified by affinity chromatography and adsorbed to reduce cross-reactivity and increase monospecificity. Antisera were evaluated against each recombinant chsp60 protein by Western blotting. Reactivity against native chsp60s was visualized by transmission electron microscopy. Initial experiments indicate that expression of the second chsp60 (encoded by groEL_2) is increased during iron limitation. The production of chsp60 antibodies in human patients is associated with damaging sequelae in chlamydial genital and ocular infections.

Chlamydia trachomatis is an obligate intracellular bacterium and the most prevalent cause of bacterial sexually transmitted disease. Genital chlamydial infections, marked by chronic, intense inflammation, can lead to genital tissue scarring and infertility and is a contributing factor to development of pelvic inflammatory disease and ectopic pregnancy. Iron is required as a cofactor for numerous highly conserved pathways, and nearly all studied organisms rely on iron for growth. In response to iron restriction, the chlamydial developmental cycle arrests at the intracellular reticulate body stage, resulting in a phenomenon termed persistence. Persistence likely plays a role in chlamydial pathogenesis through the expression of virulence factors and antigens in addition to sustaining chronic infection; however, little is known concerning how chlamydiae respond to iron limitation at the molecular level, and no systems for iron acquisition have been identified in Chlamydia. This dissertation presents an investigation into the chlamydial response to iron restriction. Chlamydial heat shock protein 60 (cHsp60) has been implicated in development of the more severe disease sequelae and has been found to increase in expression following iron restriction; however, three cHsp60 homologues were identified following the sequencing of the chlamydial genome. Here, iron restriction is shown to increase expression of cHsp60-2 but not the two other homologs, cHsp60-1 or -3. Next, in order to investigate an alternate model for restricting iron availability to chlamydiae, a cell line with inducible expression of recombinant ferroportin, a eukaryotic iron efflux protein, was examined. Lastly, 10 chlamydial proteins differentially expressed during growth in iron-restricted host cells were identified by proteomic analysis of radiolabeled proteins followed by mass spectrometry analysis; transcripts encoding 5 iron responsive proteins were examined across a timecourse of infection and revealed increased transcript levels at 18 and/or 24 hours post infection. Together, these studies have examined the molecular response of chlamydiae to reduced iron availability and have underlined the importance for pathways involved in protection against oxidative damage and adaptation to stress.

<p dir="ltr">This thesis explores the intricate mechanisms governing paracellular permeability, a process vital for maintaining homeostasis and barrier integrity in epithelial and endothelial tissues. Central to this exploration are tight junctions (TJs), multi-protein complexes that regulate substance movement between cells. The thesis begins with a comprehensive literature review synthesizing current research on how bacterial and viral proteins can enhance paracellular permeability or modulate TJs, highlighting their significance in both scientific and medical fields.</p><p dir="ltr">A focal point of the thesis is the detailed study of the <i>Listeria</i> adhesion protein (LAP), an epithelial TJ modulator (TJM). LAP’s interaction with the epithelial receptor, Hsp60, facilitates the translocation of <i>Listeria monocytogenes </i>across the epithelial barrier, presenting a novel approach for drug delivery. Addressing the limitations of current TJMs, such as poor bioavailability and toxic side effects, the thesis provides an in-depth analysis of LAP as an alternative biologics’ delivery vehicle. Utilizing a multidisciplinary research approach, the study reveals the cryo-EM structure of LAP at 2.9 Å resolution, elucidating its multimeric formation and interaction with Hsp60 through electrostatic forces, as supported by computational modeling.</p><p dir="ltr">The functional efficacy of LAP is further established through experiments demonstrating its capability to transport drug analogs across epithelial cell monolayers comparably to established TJMs. Moreover, <i>in vivo</i> studies using a mouse oral gavage model show that LAP significantly enhances the blood levels of peptide drugs without triggering inflammatory responses or harming tight junction architecture. This thesis ultimately positions the LAP-Hsp60 complex as a promising TJM candidate, offering an innovative means for enhancing oral drug delivery and maintaining epithelial barrier integrity.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Heat Shock Protein 60/10 (HSP60/10, or GroEL/ES in bacteria) chaperonin systems play a critical role in protein homeostasis through facilitating proper folding of misfolded or partially folded polypeptides that are otherwise prone to aggregation. HSP60 chaperonins are highly conserved and essential in nearly all organisms studied thus far, making them a promising target for antibiotic development. Early high-throughput screens in the Johnson lab have identified five main scaffolds that, though hit-to-lead development, have been optimized for chaperonin inhibition and antimicrobial effects. While these initial studies have shown promising evidence to support the viability of a chaperonin-targeting antibiotic strategy, it was unclear whether the conservation of human HSP60 (48% identity to bacterial GroEL) would hinder this therapeutic strategy from advancing due to potential toxicity associated with off-target inhibition of the human homolog. Additionally, while chaperonin inhibition often correlated with cytotoxicity to the various pathogens studied, there was a clear need to investigate inhibitor mechanisms to 1) verify on-target effects, and 2) guide future development of more potent and selective chaperonin-targeting antibiotic candidates. Herein, we conduct a medium-throughput screening of known bioactive molecules, approved drugs, and natural products against both bacterial GroEL and human HSP60, demonstrating that most molecules exhibited low-to-no toxicity to human cells in culture, despite being near equipotent inhibitors of human HSP60 and E. coli GroEL in our refolding assays. Thus, sequence conservation between human HSP60 and bacterial GroELs does not necessarily predict toxicity in vivo. We then investigate inhibitory mechanisms of our most well-established inhibitor series, the phenylbenzoxazole (PBZ) series, identifying three binding sites whereby PBZ molecules modulate GroEL folding and ATPase functions in a site-specific manner, predominately through its ability to interact with its co-chaperone GroES. Finally, we demonstrate that two standard of care drugs for T. brucei infections, suramin and nifurtimox, may elicit their trypanocidal effects through inhibiting HSP60. Due to structural similarities, we then screened our N-acylhydrazone (NAH) and α,β-unsaturated ketone (ABK) series of HSP60 inhibitors against T. brucei, finding that they are highly potent and selective trypanocidal agents. Together, these studies further support HSP60 as a viable drug target for antibiotic development. / 2025-07-03

Bakgrund: Myalgic Encephalomyelitis eller Chronic Fatigue Syndrome (ME/CFS) omfattar ett spektrum av olika symptom som bland annat påverkar de autonoma och neurologiska systemen, kognitiv funktion och ger immunologiska störningar med mera. De karakteristiska symptomen är oförklarlig kronisk trötthet, ansträngningsutlöst fysisk och mental uttröttbarhet Post Exertional Malaise (PEM). Trots forskning inom ett flertal områden är den underliggande molekylära orsaken bakom ME/CFS inte fastställd. Flertalet hypoteser om sjukdomsorsaken finns, varav en är att ME/CFS är en autoimmun sjukdom. Syfte: Syftet med litteraturstudien är att undersöka huruvida det finns autoimmuna aspekter i ME/CFS. Metod: Systematisk litteraturstudie utifrån vetenskapliga artiklar, publicerade 2010—2020 i databasen PubMed. Resultat: Studieresultaten är inte helt entydiga när det kommer till att påvisa autoimmuna aspekter i ME/CFS. Antikroppsstudier riktade mot neuronalt protein hos ME/CFS-patienter och behandlingar riktade mot antikroppar, immunoadsorption och rituximab, gav negativa resultat. Däremot observeras HSP60 (heat shock protein 60) antikroppar för specifika korsreaktiva epitoper i en undergrupp av ME/CFS-patienter, vilket stämmer överens med infektionsutlöst autoimmunitet. Även i de två genstudierna, HLA-association (human leucocyte antigen) och SNP (single nucleotide polymorphism) genotypning i immungener, observeras karakteristiska riskgener för autoimmun sjukdom, tydligast resultat observerades hos de patienter som har en infektionsutlöst ME/CFS. Slutsats: Trots delvis negativa resultat ges visst stöd för hypotesen då dessa indikerar autoimmuna aspekter i en undergrupp av infektionsutlöst ME/CFS. / Background: Myalgic Encephalomyelitis or Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease which is characterized by unexplained and persistent post exertional fatigue (PEM) and a myriad of symptoms related to neurological disturbance, immunological, cognitive and autonomous dysfunction. Despite biomedical research from a disparate field of expertise the pathogenesis and etiology of ME/CFS is not well-understood. Several hypotheses regarding the pathogenesis have been proposed one of which is that ME/CFS is an autoimmune disease. Aim: The purpose of the literature study is to investigate whether there are autoimmune aspects in ME/CFS. Method: A systematic literature study based on scientific articles, published 2010-2020 in the PubMed database. Results: The study results are not entirely consistent when it comes to detecting autoimmune aspects in ME/CFS. Antibody studies targeting neuronal proteins in ME/CFS-patients and antibody treatments, immunoadsorption, and rituximab yielded negative results. In contrast, HSP60 (heat shock protein 60) antibodies for specific cross-reactive epitopes are observed in a subset of ME/CFS patients, which corresponds to infection-triggered autoimmunity. The two gene studies, HLA (human leucocyte antigen) association and genotyping of SNP (single nucleotide polymorphism) in immune genes, observed characteristic risk genes for autoimmune disease. Significant results were observed in ME/CFS- patients with an infection-triggered onset. Conclusion: Despite partially negative results, some support exists for the hypothesis as results indicate autoimmune aspects in ME/CFS with an infection-triggered onset.

The overall hypothesis of this work is that the physiological microbial stress response could serve as a rapid, sensitive, and mechanistically-based indicator of process upset in biological wastewater treatment systems that receive sporadic shock loads of toxic chemicals. The microbial stress response is a set of conserved and unique biochemical mechanisms that an organism activates or induces under adverse conditions, specifically for the protection of cellular components or the repair of damaged macromolecules. Using traditional immunochemical analysis techniques, the heat shock protein, GroEL, was found to be induced in activated sludge cultures exposed to perturbations of chemicals at all concentrations tested (cadmium, pentachlorophenol, and acetone) or heat stress. As total cadmium concentrations increased above 5 mg/L, there was a significant and consistent increase in effluent volatile suspended solids concentrations from activated sludge sequencing batch reactors relative to unstressed controls, but there was no additional increase in GroEL levels. Stress proteins may serve as sensitive and rapid indicators of mixed liquor toxicity which can adversely impact treatment process performance, but GroEL may not be a good candidate protein for this purpose due to the lack of a dose/response relationship. Additionally, production of stress proteins did not explain the significant deflocculation upsets that were characteristic of many of the industrially-relevant chemicals tested, including pentachlorophenol and cadmium. Although the purpose of stress response mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. The goal of the second research phase was to determine whether the bacterial glutathione-gated, electrophile-induced potassium efflux system is responsible for deflocculation observed due to shock loads of toxic electrophilic (thiol reactive) chemicals. The results indicate significant K+ efflux from the activated sludge floc structure to the bulk liquid in response to shock loads of 1-chloro-2,4-dinitrobenzene (CDNB), N-ethylmaleimide (NEM), 2,4-dinitrotoluene (DNT), 1,4-benzoquinone (BQ), and Cd2+ to a bench-scale sequencing batch reactor (SBR) system. In most cases, the stressor chemicals caused significant deflocculation, as measured by an increase in effluent volatile suspended solids (VSS), at concentrations much less than that required to reduce the maximum specific oxygen uptake rate by 50% (IC50). This suggests that electrophile-induced activated sludge deflocculation is caused by a protective bacterial stress mechanism (as hypothesized) and that the upset event may not be detectable by aerobic respirometry. More importantly, the amount of K+ efflux appeared to correlate well with the degree of deflocculation. The transport of other cations including sodium, calcium, magnesium, iron, and aluminum, either to or from the floc structure, was negligible as compared to K+ efflux. In bench-scale SBRs, it was also determined that the K+ efflux occurred immediately (within minutes) after toxin addition and then was followed by an increase in effluent turbidity. K+ efflux and deflocculation responses were similar for bench-scale SBRs and continuous-flow reactor systems, indicating that the periods of elevated exogenous substrate levels typical in SBR systems are not required to activate electrophile-induced K+ efflux or deflocculation. This also suggests that the initial and rapid efflux of K+ immediately following electrophile addition is the factor that leads to deflocculation, not the increase in bulk liquid K+. Sphingomonas capsulata, a bacterium consistent with that found in biological wastewater treatment systems, Escherichia coli K-12, and activated sludge cultures exhibited very similar dynamic efflux/uptake/efflux responses due to the electrophilic stressors, NEM and CDNB, and the thiol reducing agent, dithiothreitol (DTT). The polyether ionophore antibiotic, nigericin, was used to artificially stimulate K+ efflux from S. capsulata and activated sludge cultures. Thus, glutathione-gated K+ efflux (GGKE) activity may cause K+ release from the cytoplasm of activated sludge bacteria into the floc structure and extracellular polymeric substances (EPS) and then diffusion-limited transport into the bulk liquid. It was not possible to resolve the effect of the GGKE system on changes in bulk liquid or floc-associated pH. However, calculations indicate that the localized K+ concentration within the floc structure immediately after chemical stress is consistent with that known to induce floc disruption as a result of KCl addition. Using alkaline phosphatase as a periplasmic marker as well as fluorescent membrane-permeable and impermeable nucleic acid stains, it was determined that a negligible amount of the K+ efflux response was due to lysis of activated sludge microorganisms. The current results are very promising and are the first to suggest that activated sludge upset (i.e. deflocculation) may be caused by a specific protective stress response in bacteria. / Ph. D.

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.