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. Author manuscript; available in PMC: 2009 Dec 11.
Published in final edited form as: Cell Host Microbe. 2008 Dec 11;4(6):519–527. doi: 10.1016/j.chom.2008.10.011

Molecular Chaperones in Pathogen Virulence: Emerging New Targets for Therapy

Len Neckers 1, Utpal Tatu 2,*
PMCID: PMC2752846  NIHMSID: NIHMS139596  PMID: 19064253

Summary

Infectious organisms have to cope with demanding and rapidly changing environments during establishment in the host. This is particularly relevant for pathogens which utilize different hosts to complete their life cycle. In addition to homeotic environmental challenges, other stressful factors, such as oxidative bursts, are often triggered in response to infection. It is not surprising that many successful pathogens have developed robust chaperone systems to conquer the stressful environments in the host. In addition to discussing ingenious ways by which pathogens have utilized chaperones, the potential of exploiting pathogen chaperones as drug targets is also discussed.

Introduction

Most pathogens encounter a drastic change in their environment during entry into the host. In addition to homeotic factors such as temperature, intracellular pathogens also face harsh conditions such as an oxidative burst triggered in response to the infection. The pathogen must cope with these environmental challenges in order to establish itself in the host. In addition to adaptation mechanisms specific to their host, elaboration of the heat shock response appears to be a common survival strategy among all pathogens. Induction of heat shock protein genes during infection has been reported for various bacteria, parasites, and viruses during an infection process. In agreement with their well studied functions, it is believed that heat shock proteins induced during infection help pathogens override unfavorable conditions found in the host. Heat shock proteins may therefore form a first line of attack and help consolidate pathogen virulence (Goulhen F. et. al, 1998, Kamiya, S. et. al., 1998, Kaneda K., et. al., 1997).

Heat shock proteins were identified for the first time in Drosophila incubated at higher temperatures (Ritossa, 1962, Tissieres A, 1974). Heat shock proteins comprise a subgroup of molecular chaperones that are induced in response to adverse environmental conditions (including but not limited to excessive heat). Molecular chaperones are both constitutive and stress-inducible, and comprise a diverse group of proteins that possess the ability to transiently assist in the non-covalent assembly or disassembly of other macromolecular structures (Hemmingsen et al., 1987). Molecular chaperones, including heat shock proteins, are essential for the survival of all three kingdoms of life, and have been shown to play key roles in the integration of cellular and organismal responses to environmental fluctuation. Given that the high cellular protein concentration (200 – 400 mg/ml) naturally favors inappropriate protein-protein interactions, leading to insolubility and protein denaturation, molecular chaperones, by transiently associating with hydrophobic surfaces of `client' proteins, are key to maintaining cellular homeostasis and a functional intracellular milieu (Ellis RJ, 2006, Minton, 2001).

Heat shock proteins are a family of structurally related proteins classified based on their molecular weights. Heat shock proteins of the classes Hsp20, Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100 have been well characterized from a large number of organisms. Proteins belonging to a particular family share extensive structural homology and functional similarity across species. Accordingly, the Hsp20 family is involved in stabilization of cytoskeletal proteins, the Hsp40 family presents substrates to the Hsp70 group of chaperones, the Hsp60, Hsp70 and Hsp90 families are involved in protein folding, and the Hsp100 family is able to disassemble protein aggregates. Chaperones that support folding of newly synthesized proteins, namely Hsp60, Hsp70 and Hsp90, have distinct mechanisms of action. While the Hsp60 family forms a double heptameric ring structure that provides a protected cavity for proteins to fold, Hsp70 and Hsp90 interact with their substrates in monomeric or dimeric forms, respectively. Hsp70 is mainly involved in supporting the early phase of protein folding while Hsp90 interacts with substrates in their near native conformations, regulating their biological activities. Most of the heat shock protein families mentioned above are constitutively synthesized but are further induced in response to stress (Bayles DO et al., 1996). Notwithstanding similarities in their structure and function across species, different organisms seem to have relied on molecular chaperones to different extents. Even prions have been shown to depend on chaperones for propagation of their infective forms (Sullivan CS et al., 2001).

There is an intense interest in exploring roles of heat shock proteins in pathogen biology. Pathogens not only utilize endogenous heat shock proteins but they also exploit host heat shock proteins. This article aims to detail our current understanding of heat shock proteins from pathogenic bacteria, viruses, protozoa and their potential roles in aiding pathogen virulence. The possibility of exploiting pathogen heat shock proteins as targets for therapy is discussed.

Bacterial heat shock proteins as virulence factors

One situation that can cause bacteria to induce heat shock proteins is after phagocytosis by the host cell where bacteria encounter stress due to phagosome acidification, oxidative burst, and phagosome fusion with lysosomes. These stimuli are capable of inducing bacterial heat shock proteins that play an important role in helping bacteria cope with stressful host environment, thereby aiding bacterial pathogenesis (Hosogi Y et al., 2005).

In bacteria, DnaK, DnaJ, GroEL and HtpG are the major heat shock protein classes. The DnaK (Hsp70) and DNA J (Hsp40) pair is involved in protein folding and is also associated with survival under stress conditions (Genevaux P et al, 2007). HtpG is an Hsp90 homolog usually present as a dimer involved in protein folding. GroEL exists in a double heptameric ring structure, presenting a protected cavity within which newly synthesized proteins can fold. All the above heat shock proteins are inducible in nature and have been implicated in helping bacteria override stressful environmental conditions (Arnold DL et al., 2007, Qoronfleh MW et al., 1998). All these chaperones share significant sequence similarities with their eukaryotic counterparts (Acharya P et al., 2007). As described below, the chaperoning roles of heat shock proteins is cleverly exploited by pathogenic bacteria for host cell pathogenesis.

DnaK is one of the best characterized bacterial chaperones. Its role in protein folding is well studied and it has also been implicated in bacterial virulence. In Brucella suis, Campylobacter jejuni and Salmonella enterica serovar Typhimurium, deletion of DnaK results in compromised growth in macrophages or inability to colonize mice (Köhler S et al., 1996, Konkel ME et al., 1998, Takaya A et al., 2004). These data support the notion that heat shock proteins are critical for survival of these bacteria in host environments.

Induction of heat shock protein genes in bacteria is either positively regulated through alternate sigma factors or negatively regulated by the transcription factor HspR (heat shock protein receptor). Manipulation of either of these factors .alters heat shock response and also influences bacterial virulence (Grophna and Ron, 2003, Kazmierczak et al, 2005). In the case of Helicobacter pylori, survival in human gastric mucosa requires it to withstand constantly fluctuating environments and low pH conditions. H. pylori responds to environmental changes by modulating a regulator of heat shock protein genes called Csr1 (cellular stress response protein1; hspR is one of the genes modulated by Csr1). Consistent with the possibility that pathogens regulate stress responses to effect virulence, an H. pylori mutant for the csr1 gene showed attenuated infection in a mouse model indicating that genes involved in stress tolerance are critical for H. pylori virulence (Barnard FM et al., 2004). However, in Mycobacterium tuberculosis, inactivation of hspR and the resulting overexpression of DnaK, causes an enhanced clearance of the bacterium in the mouse model of tuberculosis (Gupta DT et al., 2008, Smith I 2003). It appears that increased synthesis of DnaK primes the immune system early during infection possibly resulting in increased bacterial clearance. This example indicates that mere overexpression of heat shock proteins may not always confer virulence to bacteria. The timing and possibly the magnitude of expression need to be regulated for their cytoprotective effects.

One of the best known examples of the role of heat shock proteins in bacterial pathogenesis is Listeria monocytogenes infection in host macrophages. After being phagocytosed into the host, Listeria can be digested upon fusion of the phagosome with an endosomal compartment. However, the bacteria rely on a member of Hsp100 family called ClpC to overcome this consequence. Expression of ClpC in the phagosome allows Listeria to be released from the phagosome into the host cytoplasm where it undergoes multiplication (Rouquette C et al., 1998, Nair S et al., 2000). Experiments with a clpC deletion strain of Listeria show accumulation of the bacteria in phagosomes resulting in a significant reduction of bacterial load in a mouse model of infection. While the precise mechanism by which ClpC mediates bacterial exit from the phagosome is not known, it is possible that its interaction with phagosome membrane proteins may be involved. Recently a role for another member of Hsp100 family, ClpB, was shown in Francisella tularensis infection of macrophages. ClpB was essential for F. tularensis to replicate in target organs and cause pathogenesis in mice (Melbom et al, 2008). These studies clearly indicate that intracellular pathogens rely upon their endogenous chaperone machinery to survive and establish an infection within their hosts.

Besides these examples there are several reports that suggest a role for bacterial chaperones at the cell surface, as adhesins for invading the host cell or in signalling the immune system (Pizarro J et al., 2006). Both the Hsp60 and Hsp70 classes of chaperones have been implicated in this role. In addition, there are also examples where bacteria have found ways to recruit or target host chaperones to enhance bacterial growth and overcome host defense (reviewed in Henderson B at al., 2006). It is apparent that bacteria have mainly exploited their heat shock protein functions to cope with host defence mechanisms triggered in response to infections. Additional roles in invasion and multiplication have also been suggested in some cases.

Heat shock protein roles in protozoan diseases

The role of heat shock proteins as virulence factors is convincingly available in human protozoan parasites. In several pathogenic protozoa, parasite heat shock proteins have been implicated in critical stage conversion processes. In the case of Leishmania donovani, Hsp90 has been implicated in conversion of the parasite from the insect form to the mammalian form (Wiesgigl and Clos., 2001). Inhibition of Hsp90 using geldanamycin (GA) inhibits promastigote growth in culture and its overexpression overcomes this growth inhibition. In the case of Trypanosoma cruzi, Hsp90 has also been implicated in proliferation of epimastigotes and its inhibition abrogates conversion of blood form trypomastigotes into epimastigotes (Graefe SE et al., 2002). More recently a similar role for Hsp90 was demonstrated in Toxoplasma gondii wherein its development from the bradyzoite to tachyzoite stage was inhibited and entry of T. gondii into the host was also compromised upon inhibition of its Hsp90 (Echeverria PC et al., 2005, Ahn HJ et al., 2003). In Plasmodium falciparum, parasitic Hsp90 was essential for P. falciparum growth in human erythrocytes. Hsp90 inhibition using GA abrogated P. falciparum asexual development in human erythrocytes (Banumathy et al., 2003). A similar role for Hsp90 in Eimeria and Babesia parasites has been demonstrated (Peroval M et al., 2006, . The use of Hsp90 to control important developmental transitions in the host appears to be conserved in protozoa.

It is necessary to understand the organization of Hsp90 in the cell to appreciate how a chaperone may influence complex cellular processes such as stage development. In all eukaryotic cells, cytosolic Hsp90 exists as a multi-chaperone complex (Pratt and Toft, 2003). In this complex, a dimeric form of Hsp90 is usually associated with Hsp70 and other co-chaperones such as Hop (Hsp70-organizing protein), p23, Aha1 and cyclophilin. These co-chaperones modulate the weak ATPase activity associated with Hsp90 and thereby regulate client protein binding to Hsp90. Hsp90 chaperones a group of client proteins involved in signal transduction and cell cycle control. The clients include protein kinases and transcription factors that respond to environmental stimuli to bring about cell cycle regulation or signal transduction (Pratt and Toft, 2003). Such ability of Hsp90 to link environmental changes to changes in gene expression and cell cycle control is shrewdly exploited by pathogens to potentiate their virulence.

It appears that parasites have utilized heat shock proteins to sense environmental cues in the host and to signal when appropriate developmental transitions in the parasite should occur. Indeed, many effectors of signaling machinery in intracellular protozoan parasites such as Plasmodium are conserved with their hosts (Vaid and Sharma, 2006). By conserving the signal transduction components of the Hsp90 multi-chaperone complex, these intracellular parasites seem to have mimicked the ability of their host to respond to environmental signals such as changes in temperature (Denis M. et. al, 1988, Hartson SD, et. al., 1996).

Apart from Hsp90, heat shock proteins of the class Hsp60, Hsp70 and Hsp100 have also been implicated in the pathogenesis of protozoan infections. Hsp100 is critical for intracellular parasite survival and contributes to the infectivity of Leishmania major (Hubel et al., 1997, Krobitsch & Clos, 1999, Reiling L et al., 2006). Hsp100−/− mutants of L. major show reduced survival in macrophage and attenuated growth in BALB/c mice. Similarly an increase in infectivity is associated with increased levels of heat shock proteins in L. braziliensis (Smejkal RM et al., 1988). In T. gondii, Hsp70 plays an important role in evading host proinflammatory responses and thereby contributes to its virulence (Dobbin et al, 2002). Thus, in addition to utilizing their conventional roles in stress tolerance, parasites have exploited heat shock proteins to fine tune their growth and development according to conditions prevailing inside the host.

Most recently, transcriptome analysis of malarial parasites isolated from patients has revealed a significant correlation between upregulation of genes coding for major heat shock protein families of the parasite and the clinical outcome of infection (Daily JP et al., 2007). Importantly, patients showing similar clinical characteristics of high interleukin levels and febrile conditions had Plasmodium parasites showing a similar profile of heat shock proteins upregulated. In an independent group of patients showing an intermediate clinical manifestations, a cohort of functionally related, cytoplasmic heat shock proteins were significantly upregulated in their Plasmodium parasites. This included all the components of the Hsp90 multi-chaperone complex described above. The results suggest that heat shock proteins may participate in shaping the clinical course of malaria. It would be interesting to see if the heat shock protein profiles seen at transcriptome level also translate at the level of the proteome and if there are any correlations with the severity of disease. Thus, in addition to exploiting heat shock proteins to counter environmental challenges unleashed by the host, protozoan parasites have also evolved chaperone dependent mechanisms to sense and utilize changes in the host environment as developmental cues to coordinate their growth.

Heat shock proteins in virus infections

While most viruses themselves do not code for heat shock proteins, they have instead evolved to rely on the host chaperone machinery for their folding and assembly needs much as viruses do for many other processes. It is well known that animal viruses such as influenza and vesicular stomatitis virus (VSV), code for envelope proteins such as hemagglutinin and VSV G, whose glycosylation, folding, assembly and transport are dependent on host chaperones. In fact, many of the cellular mechanisms involving protein folding and assembly were first discovered using viral glycoproteins as models (Tatu U et al., 1995, Tatu U et al., 1997).

In addition to chaperones of the secretory pathway, many viruses also utilize cytosolic chaperones for modulating their protein function. Hsp40, Hsp70 and Hsp90 classes have been implicated in viral protein dynamics in the cell. There are also examples wherein viruses pack host-chaperones within their capsid structure before being released out of the cell (Gurer C et al., 2004). These molecules are later utilized in the invasion of new cells.

Viruses often code for co-chaperones of the Hsp40 class that are capable of recruiting Hsp70 chaperones of the host. The classic example of host-chaperone recruitment by viruses is in case of a polyoma virus SV40. Tumor antigen (T antigen) of this virus harbors an Hsp40-like J domain, capable of interacting with Hsp70. The T antigen recruits host Hsp70 into a virus infected cell to inactivate the retinoblastoma tumor supressor gene product (pRb; Retinoblastoma protein) and thereby plays an essential role in host cell transformation by the virus (Sullivan CS et al., 2002).

Host cytoplasmic Hsp70 has been utilized by many viruses during their multiplication in the host. Recently, human immunodeficiency virus (HIV) was shown to incorporate host Hsp70 in the virion core (Gurer C et al., 2005). Inhibition of the virion incorporated Hsp70 ATPase activity disrupted the core conformation of Hsp70 and reduced virus infectivity. In the case of hepatitis B virus, host Hsp90 is utilized to support ribonucleoprotein assembly between viral reverse transcriptase and ε RNA of the virus (Jianming Hu et al., 1996). A role for host Hsp90 was also shown in vaccinia, hepatitis C and negative strand RNA viruses such as VSV G (Hung JJ et al., 2002, Nakagawa S et al., 2007, Connor JH et al., 2007). While the mechanistic role of host Hsp90 has not been studied in these viruses, its inhibition disrupted virus growth in culture and in a mouse model. More recently host Hsp90 was shown to be required for capsid assembly in poliovirus, rhinovirus and coxsackie virus (Geller R et al, 2007). Pharmacological inhibition of host Hsp90 inhibited replication of these viruses in cell culture and in animal models (Geller R et al, 2007). In another example, host Hsp40 is utilized by HIV to activate its nef (negative factor) gene required for viral infectivity and replication (Kumar M et al., 2005). In this case, HIV infection also caused induction of host Hsp40 to be utilized by the virus to support viral gene transcription. Nef association with host Hsp40 promoted its nuclear translocation and association with the CDK9 transcription complex. Thus, despite lack of their own heat shock protein coding genes, viruses have developed novel mechanisms to recruit heat shock proteins of the host to support processes ranging from virus entry, protein folding, trafficking, virus multiplication and assembly inside the host.

Drug resistance

Microbes are rapidly acquiring resistance to all the existing drugs and development of new drugs is a slow process. Antimicrobial drug resistance is therefore a major problem in the treatment of infectious diseases such as malaria, tuberculosis, acquired immunodeficiency syndrome (AIDS), and bacterial infections as well as cancer. A large number of patient deaths result from the failure of therapy due to drug resistant forms of pathogens. Therefore overcoming drug resistance needs to be pursued with a sense of urgency. A recent report in fungal pathogens linked Hsp90 function to evolution of resistance to antimicrobial drugs (Cowen LE et al., 2005). Inhibiting heat shock protein function therefore presents a possible new approach to overcoming drug resistance.

A study on the development of resistance to azoles in Saccharomyces cerevisiae has shown that appearance of drug resistance was dependent on Hsp90 function (Cowen LE et al., 2006). Azoles are commonly used antifungal drugs that inhibit Erg11 (lanosterol 14-alpha demethylase) and block biosynthesis of ergosterol, an important component of fungal membranes. Inhibition of Erg11 results in accumulation of intermediates that are toxic and cause membrane stress. High concentrations of Hsp90 in an engineered strain of S. cerevisiae promoted evolution of rapid drug resistance under an acute selection regime with a high concentration of drug, while Hsp90 inhibition precluded development of drug resistant phenotypes. Azole resistance arose due to a mutation in Erg3 resulting in inhibition in accumulation of toxic intermediates. The study also showed that Hsp90-dependent emergence of drug resistance was mediated by an Hsp90 client called calcineurin (a calcium dependent protein phosphatase) and its downstream effector Crz1 (calcineurin responsive zinc finger transcription factor). In addition, Hsp90 dependent calcineurin activation is known to be involved in coping with stressors including membrane stress due to azoles (Cowen, LE, et. al., 2006).

It is important to mention here that Hsp90 has been implicated in regulating environmental influences on phenotypes arising from cryptic genetic variants present in individual genomes (Rutherford and Lindquist, 1998). Such phenotypes are often stabilized and enriched at the genetic level, depending on environmental selection pressures and their fitness. Hsp90 enables cellular stress responses crucial for resistance to evolve and to be maintained. It is conceivable that additional Hsp90-dependent pathogenic traits may manifest in response to stressful host environment and promote pathogen virulence.

Inhibiting Hsp90 function therefore provides a possible new approach to overcome drug resistance and counter parasite virulence. Indeed, Leishmania Hsp83 (a homolog of Hsp90) has been implicated in responses to antimonial drug therapy in drug resistant clinical isolates (Vergnes B et al, 2007). A proof of principle study was performed in vitro where GA was used to inhibit Hsp90 to overcome azole resistance in fungi (Cowen LE et al., 2005). However, new Hsp90 inhibitors will be required to overcome the limitations of GA such as its hepatotoxicity in order to be useful for clinical treatment in real life.

In all, the examples discussed above make a compelling case for the involvement of molecular chaperones in development of antimicrobial drug resistance. Though limited in number, available examples do provide a mechanistic basis for chaperone dependent drug resistance that is likely to be utilized by other infectious disease causing microbes as well.

Therapeutic approaches targeted at heat shock proteins

As a corollary to their potential roles in parasite virulence discussed above, molecular chaperones have been considered as targets for therapeutic interventions. There are examples of natural small molecules as well as proteins produced by microorganisms that can bind and inhibit chaperone proteins. By producing specific inhibitors, microorganisms have cleverly targeted the critical nature of chaperone function as a strategy to survive in highly competitive natural environments.

At a first glance, the possibility of using chaperones as drug targets does not seem attractive because of the high degree of homology seen between chaperones from different sources. Despite overall structural similarity between human and pathogen chaperones, they display different dependencies on chaperone supported pathways and may therefore exhibit differences in sensitivities to their inhibition.

The best known example of an inhibitor of chaperone function is GA, an ansamycin ring containing molecule produced by Streptomyces hygroscopicus (Deboer C et al., 1976). GA is a specific inhibitor of Hsp90. By competing for ATP binding, GA inhibits the ATPase activity of Hsp90 and thereby prevents client protein cycling and maturation (Figure 1, Grenert JP et al., 1997). The molecule exhibits a high degree of specificity towards Hsp90 as its Bergerat-fold ATP binding cavity is shared by a limited number of ATP-binding proteins (Neckers L, et al, 1999). An obvious outcome of GA binding to Hsp90 is degradation of its client proteins that include protein kinases and transcription factors regulating cell cycle control and signal transduction pathways (Figure 1). The essential nature of these clients results in inhibition of cell growth in most systems (Neckers L, 2002). The extent of growth inhibition conferred by GA varies depending on the importance of Hsp90-dependent pathways in different systems.

Figure 1. ATPase cycle of Hsp90 drives client protein maturation.

Figure 1

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(A) Dimeric Hsp90 bound to its client protein. ATP binding and hydrolysis results in conformational changes in Hsp90 that drive client protein maturation and release. (B) GA competes for ATP binding site on Hsp90 N terminal domain. GA binding to Hsp90 dissociates the client protein resulting in its proteasomal degradation. N, N terminal domain; M, middle domain; C, C terminal domain; GA, geldanamycin; U and Ub, ubiquitin.

Interestingly, the inhibitors of Hsp90 discussed above, have also found use as an anti-cancer drug because of the critical reliance of cancer progression on Hsp90 dependent client proteins (Neckers L, 2002, Neckers L et al., 1999). GA derivatives have gone through phase II clinical trials towards application in cancer therapy. As detailed above, protozoa causing infections in humans have critically relied on Hsp90 function for their development in mammalian hosts. Indeed Hsp90 counterparts from Leishmania, Trypanosoma, Plasmodium and Toxoplasma parasites have been proposed as potential drug targets and preliminary data is available from various labs to support this possibility (Wiesgigl and Clos., 2001, Graefe SE et al., 2002, Echeverrhavia PC et al., 2005, Banumathy G et al., 2003). Hsp90 inhibitors have also been suggested as therapeutics for picornavirus infections such as polio (Geller R et al, 2007). Interestingly, a monoclonal antibody directed at Hsp90 of Candida albicans has also been developed by NewTec Pharma PLC as a novel approach to treat candidiasis (Matthews RC., et al, 2003).

It is also possible that pathogen chaperones, in spite of their close similarity to host proteins (Kumar R et al., 2007), could be specifically targeted with novel small molecules. For example, although the Hsp90 ortholog in the nematode Caenorhabditis elegans does not interact with and is not inhibited by GA, Hsp90 in the filarial nematode Brugia pahangi is efficiently inhibited by the drug, which effectively sterilizes the worm (David et al., 2003; Devaney et al., 2005). This is the case despite a high degree of conservation between the two nematode Hsp90 sequences. Similar divergent and unexpected sensitivity to Hsp90 inhibitors has also been observed when human Hsp90 is expressed as the sole Hsp90 isoform in yeast, or when non-inactivating point mutations are introduced into the Hsp82 isoform of yeast Hsp90 (Piper et al., 2003a; Piper et al., 2003b). Further, it is becoming apparent that distinct cell-specific post-translational modifications to Hsp90, including acetylation and phosphorylation of unique chaperone residues, alter interaction with and affinity for Hsp90 inhibitors (Scroggins et al., 2007a; Scroggins & Neckers, 2007b). These findings suggest that a high-throughput screen to identify pathogen-specific Hsp inhibitors is feasible and may prove to be a fruitful approach to the development of novel drugs.

Conclusions

In summary, microbial pathogens have successfully exploited molecular chaperones to overcome challenges confronted in the inhospitable host cell environment. Processes ranging from pathogen entry, establishment and multiplication have all been shown to exploit chaperone proteins to facilitate pathogenesis. While detailed mechanisms are available for some, others are known only at a descriptive level. A summary of representative examples of heat shock protein involvement in microbial virulence is shown in Figure 2 and Table 1. The following general themes emerge from the evidences implicating chaperones in supporting parasite virulence mechanisms. (1) Growth of pathogenic microorganisms in their host is often marked by induction of their heat shock protein genes. (2) Specific heat shock protein gene deletions in pathogens result in attenuation of their growth within the host. (3) Among different families of heat shock proteins, Hsp60 (bacteria), Hsp70 (bacteria and viruses) and Hsp90 (protozoa and viruses) have been most commonly implicated in microbial virulence. Among organellar heat shock proteins of protozoan parasites, cytoplasmic heat shock proteins were most commonly involved in aiding survival in the host. (4) Owing to its involvement in responding to environmental stimuli, cytoplasmic Hsp90 has been most commonly and strategically implicated in microbial pathogenesis. (5) Therapeutic strategies targeting heat shock proteins have been successfully applied in controlling fungal infections. Inhibition of heat shock protein function using small molecule inhibitors is able to overcome infection process in cell culture as well as in animal models of the disease.

Figure 2. Imperius, Cruciatus and Avada Kedavra: Molecular Chaperones in Disease Pathogenesis.

Figure 2

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Just as Lord Voldemort and his followers use Imperius, Cruciatus and Avada Kedavra curses to overpower their opponents in Harry Potter series, pathogens often use molecular chaperones to conquer their hosts. Intracellular pathogens of various classes from bacteria to protozoa, employ molecular chaperones to invade host cells, establish infection, respond to environmental stimuli and counter the effects of inhibitory drugs. Chaperones may be of host or pathogenic origin. Viruses typically exploit human chaperones to support viral transcription and replication. Bacteria and protozoa encode their own chaperones necessary for infection. (1) Invasion of host cells by Salmonella enterica, Actinobacillus actinomycetemcomitans, Listeria monocytogenes rely heavily on chaperones encoded by these organisms (Takaya A et al., 2004, Zhang L et al., 2001, Nair S et al., 2000) . (2, 3, 4) Following invasion, pathogens encounter environmental stress in the form of reactive oxygen species, acidic environment in phagosomes and heat stress in the form of fever in the host. Different pathogens recruit different chaperones to counter this onslaught and establish infection within the host (Wiesgigl M and Clos l., 2001, Graefe SE et al., 2002, Echeverrhavia PC et al., 2005, Banumathy G et al., 2003, Singh VK et al., 2007, Basak C et al.,2005). (5) Hsp100 is involved in lysosome fusion of Listeria monocytogenes (Nair S et al., 2000). (6) Furthermore, pathogenic chaperones have been implicated in development of resistance to inhibitory drugs (Cowen LE et al., 2006, Cowen LE et al., 2005). (7) Growth and multiplication of certain pathogens such as Francisella tularensis requires chaperones (Melbom KL et al., 2008). (8) HIV even exploits the host endoplasmic reticulum (ER)-chaperone machinery in order to export its surface protein gp120 to the infected cell plasma membrane (Otteken A et al.,1996). (9) Viruses such as HIV and the protozoan parasite Plasmodium falciparum have been shown to recruit host chaperones to establish a host environment that facilitates infection (Silva Ed et al., 1994, Kumar M et al., 2005, Sullivan CS et al., 2002). Pathogens therefore utilize molecular chaperones in facing challenges that they encounter in their hosts as well as in exploiting host resources to their own ends.

Table 1. Heat shock proteins involved in pathogenicity.

Representative examples of the roles of Hsp40, Hsp70, Hsp90 and Hsp100 in supporting virulence in viral, bacterial and protozoan diseases.

Chaperone Organism Disease Function Reference
Hsp100 (ClpC) Listeria monocytogenes Listeriosis Promotes early bacterial escape from phagosomal compartment of macrophages Nair S et al., 2000
Hsp100 (ClpB) Francisella tularensis Tularemia ClpB required for multiplication in host organs Melborn KL et al., 2008
Hsp90 Leishmania donovani Trypanosoma cruzi Plasmodium falciparum Leishmaniasis Chagas' disease Malaria Parasite Hsp90 functions as a sensor of environmental cues Wiesgigl M et al., 2001 Graefe SE et al., 2002 Echeverrhavia PC et al., 2005 Banumathy G et al., 2003
Hsp90 Picorna viridae Polio Foot and mouth disease Host Hsp90 required for capsid assembly Geller R et al, 2007
Hsp70 HIV AIDS Virus encapsidated host Hsp70 necessary for infectivity Gurer C et al., 2005
Hsp70 Toxoplasma gondii Toxoplasmosis Hsp70 involved in modulation of NF-κB and NOa Dobbin et al, 2002
Hsp40 SV40 virusa Tumorigenesis Host Hsp70 recruitment and cell transformation Sullivan CS et al., 2002
Hsp40 HIV AIDS Host Hsp40 involved in nef gene activation and virus replication Kumar M et al., 2005
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a

NF-κB, nuclear factor-kappa B; NO, nitric oxide; SV40, simian virus 40.

Clearly chaperone involvement in virulence is still a nascent field and several leads need to be followed up. As stated above, Hsp90 is most commonly implicated in disease progression and pathogenesis of microbial infections. It would be desirable to develop small molecule inhibitors targeted specifically at Hsp90 of microbial pathogens. About 50% identity is found at the level of the primary structure among Hsp90s from human and microbial pathogens (Acharya P et al., 2007). It appears possible to exploit differences in Hsp90 sequences between Hsp90's from human and microbial pathogens to tailor molecules specific to the pathogen. Importantly, Hsp90 has been linked to emergence of new traits such as development of drug resistance and its inhibition may therefore provide a solution to the increasing appearance of drug resistant pathogens. Attention also needs to be given to the possibility of finding chaperone targets unique to the pathogen, e.g. certain Hsp40s unique to the malarial parasite (Pavithra et al, 2007). Our growing awareness about roles of molecular chaperones in cellular processes such as protein biogenesis, signal transduction, development, evolution and their clever utilization by microbial pathogens in virulence raises the possibility of their exploitation as drug targets to treat ever increasing human infectious diseases. Indeed chaperones are already viewed as potential targets in many human ailments such as cancer and neorodegenrative disorders. Harnessing the existing knowledge about the chemistry, biology and clinical trials of chaperone inhibitors presents an excellent opportunity to explore their potential in the treatment of infectious diseases.

Acknowledgements

UT would like to thank Ms. Pragyan Acharya and Ms. Rani Pallavi for carefully reading the manuscript and Ms. Syama Chandran for her technical assistance. Financial support from Department of Biotechnology and Council of Scientific and Industrial Research, New Delhi, India is gratefully acknowledged. Authors may not have been able to cover many important references due to space limitations.

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