**5. Hsps in** *M. tuberculosis*

260 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

1992). Hsp70 proteins in the endoplasmic reticulum are involved in two distinct chaperone functions in the normal cell. In the first, the Hsp70 family chaperone transfers the newly synthesized, unfolded protein to Hsp60 family of chaperonins, leading to eventual folding of the proteins. In the second case, Hsp70 chaperones carry proteins to different cellular compartments for the proper folding of the proteins (Kiang & Tsokos, 1998, Shi & Thomas,

Chaperonins are ring shaped proteins involved in promoting the ATP dependent folding of proteins under normal as well as under stress conditions. GroE machinery is the most prominent chaperonin in bacteria (Horwich et al., 2006). It consists of 14 GroEL subunits arranged in a cylinder of two heptameric rings, which is further attached to a heptameric ring of GroES (Horwich et al., 2006). GroE can bind to several different types of non-native proteins. The non-native protein is encapsulated in the GroE cylinder. GroEL internalizes the protein for the length of ATP hydrolysis cycle, during which the protein can refold to its native state (Viitanen et al., 1992). The closely related proteins in the mitochondria are called

Small Hsps (sHsps) are the most poorly conserved group among Hsps. Their most common trait is an α-crystallin domain. The most prominent member is the eye lens protein αcrystallin or Acr (Horwitz, 2003). sHsps are ATP-independent chaperones that form a large oligomeric structure often composed of 24 subunits. sHsps interact with partially folded targeted proteins to prevent their aggregation under stress conditions (Haslbeck et al., 2005). sHsp are also shown to be important in protecting the cell against the numerous injuries like

Hsps play a central role in managing the damaged or aggregated proteins inside the cells. They have been linked to the virulence of several pathogenic microbes. *Candida albicans* expresses a bonafide heat shock response that is regulated by the evolutionarily conserved, essential heat shock transcription factor Hsf1. Hsf1 is thought to play a fundamental role in thermal homeostasis, adjusting the levels of essential chaperones to changes in growth temperature (Brown et al., 2010). In *Plasmodium falciparum* heat shock protein 70 is thought to play an essential role in parasite survival and virulence inside the host; Hsp70 is also being tried as a target for designing potential anti-malarial drugs (Cockburn et al., 2010). *Histoplasma capsulatum* is the causative agent of histoplasmosis in humans. A 62 kDa Hsp (Hsp60) of *H. capsulatum* is an immunodominant antigen which has been shown to play an important role in the adaptation of the fungus to temperature stress (Guimaraes et al., 2010). *Staphylococcus aureus* and *Staphylococcus epidermidis* can cause serious chronic infections in humans. An important factor involved in the pathogenesis of *S. aureus* is its ability to be internalized by phagocytes thereby evading the host immune system. Heat shock cognate protein, Hsc70 was identified as playing an important role in the internalization mechanism of *S. aureus* (Hirschhausen et al., 2010). ClpB gene from *Enterococcus faecalis* is linked to thermotolerance and virulence of the bacteria (de Oliveira et al., 2010). The Clp proteases appear to be critical for cell development in *Caulobacter crescentu*, and stress induction in *Bacillus subtilis* (Gerth et al., 2004)*.* ClpC has been linked to the tight regulation of virulence genes in *Listeria monocytogenes*; it has been shown to be required for adhesion and invasion of the pathogen (Nair et al., 2000). ClpC has also been shown to be important for the

heat stress, oxidative stress and apoptosis inducing factors (Arrigo, 1998).

**4. Hsps and virulence in pathogenic microorganisms** 

1992).

as Hsp60 and Hsp10.

The ability of *M. tuberculosis* to survive under oxidative stress *in vivo* is an important aspect of its pathogenesis. Heat shock proteins are essential molecular chaperones for maintaining cellular functions during normal as well as stress conditions. The heat shock proteins also play a role in antigen presentation, and activation of lymphocytes and macrophages (Tsuchiya et al., 2009). The virulence of mycobacterium is dependent upon multiple genes that are expressed for the successful survival of the pathogen inside the macrophage. Expression of many heat shock proteins have been shown to increase under stress conditions in *M. tuberculosis* (Monahan et al., 2001; Sherman et al., 2001; Stewart et al., 2002; Voskuil et al., 2004). Proteome analysis of *M. tuberculosis* showed increased expression of Hsps such as 16 kDa α-crystallin (HspX), GroEL-1 and GroEL-2 inside macrophages. Hypoxia and starvation induce stationary phase in *M. tuberculosis*, under these conditions there is increased expression of hspX and acr2 (Sherman et al., 2001; Voskuil et al., 2004). Exposure of *M. tuberculosis* to heat shock induced the expression of hsp70 regulon, groEL, groES and acr protein (Stewart et al., 2002). The deletion of HspR, a repressor of Hsp70 proteins in *M. tuberculosis* has important impact on virulence. A HspR deletion mutant overexpressed Hsp70 proteins, and was fully virulent in the initial stages of infection; however the ability of the bacteria to establish a chronic infection was impaired as compared to the wild type (Stewart et al., 2001). The expression of Hsp65 and Hsp71 of *M. bovis* was increased under heat shock (Patel et al., 1991).

The synthesis of Hsps is increased after infection, some of which are immunodominant antigens in *M. tuberculosis* and *M. leprae* (Young et al., 1988). Hsp70 is an immunodominant antigen in *M. tuberculosis, M. leprae, Leishmania donovani, Plasmodium faciparum and Trypanosoma cruzi* (Kaufmann, 1994; Kiang & Tsokos, 1998). The heat shock protein, DnaK and many other proteins show increased expression during survival in carbon-starved stationary phase in *Mycobacterium smegmatis* (Blokpoel et al., 2005). In addition to a significant role in immune response, Hsps may also play a direct role in the virulence of *M. tuberculosis.* The over-expression of Hsps in *M. tuberculosis* leads to a better survival at higher temperature as compared to the wild type because of the protective effect of higher levels of Hsp (Stewart et al., 2001). Heat shock protein 22.5 (Hsp22.5) is a member of heat shock regulon which was shown to be activated under stress conditions, including survival in macrophages and during the late phase of chronic tuberculosis in murine lungs (Abomoelak et al., 2010). Deletion of Hsp22.5 resulted in the modulation of transcription of important genes like dormancy regulon, ATP synthesis, respiration, protein synthesis, and lipid metabolism (Abomoelak et al., 2010). Heat shock in *M. tuberculosis* has been shown to induce the expression of Acr2, a novel member of the α-crystallin family of molecular

Heat Shock Proteins in *Mycobacterium tuberculosis*:

**6. Hsps as antigens in** *M. tuberculosis* 

than inflammatory potential (Motta et al., 2007).

(Sherrid et al., 2010).

Involvement in Survival and Virulence of the Pathogen 263

dairy and sheep industries showed increased expression of ClpB gene during infection (Hughes et al., 2007). ClpB, of *M. bovis* BCG showed reactivity with sera of TB patients suggesting it to be an antigen target of the human immune response to mycobacteria (Bona et al., 1997). There are specific transcription factors that are involved in the regulation as well as transcription of Hsps during different conditions. ClgR, a clp gene regulator of *M. tuberculosis* activates the transcription of at least ten genes, including four that encode protease systems ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c, and three that encode chaperones Acr2, ClpB and the chaperonin Rv3269 (Estorninho et al., 2010). This transcriptional activation and regulatory function of ClgR is very important in the replication of bacteria inside the macrophages. It has been shown that ClgR deficient *M. tuberculosis* is not able to resist the pH inside macrophage post infection (Estorninho et al., 2010). The mechanism by which mycobacteria return to a replicating state after a nonreplicating state, when exposed to low oxygen tension conditions is not clearly understood. ClgR is also implicated in the resumption of replicating state after hypoxia in *M. tuberculosis*

Hsps may be released extracellularly upon necrotic cell death or independent of cell death. The mechanism of the release of Hsps is not clear (Tsan & Gao, 2004). The human as well bacterial Hsps stimulate immune response. The bacterial Hsps might modulate immunity by rapidly and directly increasing cytokine production in macrophages. T cells reacting to Hsp65 appear to play an important role in the control of *M. leprae* infection (de la Barrera et al., 1995). Hsp65 directly activates monocytes during mycobacterial infection. It leads to the production of TNF (tumor necrosis factor), IL-6 and IL-8. These cytokines are important in developing antigen specific T-cell mediated host immunity (Friedland et al., 1993). The murine intraepithelial lymphocytes (IEL), when exposed to soluble extract from *M. tuberculosis* showed elevated expression of IL-3, interferon-γ and IL-6 (Mendez-Samperio et al., 1995). Peripheral blood mononuclear cells (PBMC) from TB patients showed proliferative response to the Hsp65 (Mendez-Samperio et al., 1995). The *in vitro* immune responses to *M. tuberculosis* Hsp65 were checked in TB patients, and their PBMC showed high IFN-γ levels (Antas et al., 2005). When guinea pigs were vaccinated or infected with *M. bovis* (BCG) and virulent *M. tuberculosis*, cellular and humoral immune responses to mycobacterial stress proteins Hsp65 and Hsp70 were detected (Bartow & McMurray, 1997). The C-terminal portion of heat shock protein Hsp70 was shown to be responsible for stimulating Th1-polarizing cytokines in human monocytes to produce IL-12, TNF-α, NO, and C-C chemokines (Wang et al., 2002). Hsp70 induces the expression of IL-10 and inhibits T-cell proliferation *in vitro*. Hsp70 appears to have immunosuppressive properties rather

Hsp71 and Hsp65 are the major active components of the soluble extract of *M. tuberculosis.*  Murine IELs were induced to divide and to secrete cytokines by Hsp71 and Hsp65 (Beagley et al., 1993). *M. tuberculosis* Hsp70, *M. leprae* Hsp65, and *M. bovis* BCG Hsp65 increased the levels of cytokines IL-1α, IL-1β, IL-6, TNFα, and GMCSF in macrophages (Retzlaff et al., 1994). *M. tuberculosis* contains multiple genes encoding Cpn 60 proteins, and these chaperonins have been involved in directly activating human monocytes and vascular

chaperones (Wilkinson et al., 2005). The expression of acr2 increased within 1 h after infection of monocytes or macrophages. A deletion mutant (Δacr2) was unimpaired in log phase growth and persisted in IFN-γ-activated human macrophages (Wilkinson et al., 2005). GroES, also known as cpn.10 is found as a major constituent in the culture filtrate of *M. tuberculosis*, suggesting that it is exposed to the intraphagosomal milieu; it may be playing an important role in the survival of bacteria inside the phagosome (Sonnenberg & Belisle, 1997). Wayne and Sohaskey (2001) suggested that the decreased effectiveness of rifampin in the non-replicative state could be because of the stabilizing effect of chaperonin. Thus, a combination therapy of rifampin and a chaperonin inhibitor has the potential to shorten the therapeutic regimen. CD43, a large sialylated glycoprotein found on the surface of haematopoietic cells is involved in efficient macrophage binding and immunological responsiveness to *M. tuberculosis. M. tuberculosis* employs Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70) as an adhesin that binds CD43 on the macrophage surface (Hickey et al., 2010). The crystal structure of the chaperonin 60 of *M. tuberculosis*, also called Hsp65 or chaperonin 60.2 has been solved (Qamra & Mande, 2004). Another *M. tuberculosis* small heat shock protein 16.3 (Hsp16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection and its expression is increased in response to stress (Valdez et al., 2002). It contains the core 'α-crystallin' domain found in all sHsps and protects against protein aggregation *in vitro* (Valdez et al., 2002). Protein phosphorylation is frequently used by organisms to adjust to environmental variations. Hsp16.3 and Hsp70 are immunodominant proteins synthesized during the *M. tuberculosis* infection. It was shown that these Hsps possess autophosphorylation activity (Preneta et al., 2004). *M. tuberculosis* genome has revealed the presence of heat shock proteins ClpP1, ClpP2, ClpC1, ClpX and ClpC2. The ClpC1 of *M. tuberculosis* has been shown to have an inherent ATPase activity, and to prevent protein aggregation as a chaperone in the absence of any adaptor protein (Kar et al., 2008). *M. tuberculosis* ClpC1 has also been shown to interact with ClpP2 (Singh et al., 2006). *M. tuberculosis* ClpC1 has been shown to interact with ResA, an anti sigma factor which is degraded by ClpC1P2 protease *in vitro* (Barik et al., 2010). Knockdown of ClpC1 in *M. smegmatis* and *M. tuberculosis* showed inhibition of RseA degradation indicating a regulatory role of Clp proteins in *M. tuberculosis* (Barik et al., 2010). ClpX is predicted to be essential for *in vivo* survival and pathogenicity and is conserved in *M. tuberculosis, M. leprae, M. bovis* and *M. avium paratuberculosis* (Ribeiro-Guimaraes & Pessolani, 2007). ClpX of *M. tuberculosis* was not able to substitute ClpC1 in ClpC1P2 protease complex (Barik et al., 2010). Knockdown of ClpX in *M. smegmatis* did not prevent the degradation of RseA indicating that ClpP2 does not associate with ClpX for its proteolytic activity (Barik et al., 2010). Leprosy and tuberculosis patients with active disease had shown the presence of antibodies recognizing ClpC in dot ELISA (Misra et al., 1996). The expression of ClpX was found to be upregulated in *M. tuberculosis* upon macrophage infection (Dziedzic et al., 2010). FtsZ is a protein known to assemble at the midcell division site in the form of a Z-ring. It is crucial for initiation of the cell division process in eubacteria. ClpX has been shown to interact with FtsZ in *M. tuberculosis* (Dziedzic et al., 2010). The crystal structure of *M. tuberculosis* caseinolytic protease, ClpP1 showed a disordered conformation of the residues in the catalytic triad, which makes the protein inactive (Ingvarsson et al., 2007). ClpP of *M. tuberculosis* has been studied as a target for drug designing (Tiwari et al., 2010). *M. avium*, the causative agent of paratuberculosis (Johne's disease) and an economic problem for beef,

chaperones (Wilkinson et al., 2005). The expression of acr2 increased within 1 h after infection of monocytes or macrophages. A deletion mutant (Δacr2) was unimpaired in log phase growth and persisted in IFN-γ-activated human macrophages (Wilkinson et al., 2005). GroES, also known as cpn.10 is found as a major constituent in the culture filtrate of *M. tuberculosis*, suggesting that it is exposed to the intraphagosomal milieu; it may be playing an important role in the survival of bacteria inside the phagosome (Sonnenberg & Belisle, 1997). Wayne and Sohaskey (2001) suggested that the decreased effectiveness of rifampin in the non-replicative state could be because of the stabilizing effect of chaperonin. Thus, a combination therapy of rifampin and a chaperonin inhibitor has the potential to shorten the therapeutic regimen. CD43, a large sialylated glycoprotein found on the surface of haematopoietic cells is involved in efficient macrophage binding and immunological responsiveness to *M. tuberculosis. M. tuberculosis* employs Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70) as an adhesin that binds CD43 on the macrophage surface (Hickey et al., 2010). The crystal structure of the chaperonin 60 of *M. tuberculosis*, also called Hsp65 or chaperonin 60.2 has been solved (Qamra & Mande, 2004). Another *M. tuberculosis* small heat shock protein 16.3 (Hsp16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection and its expression is increased in response to stress (Valdez et al., 2002). It contains the core 'α-crystallin' domain found in all sHsps and protects against protein aggregation *in vitro* (Valdez et al., 2002). Protein phosphorylation is frequently used by organisms to adjust to environmental variations. Hsp16.3 and Hsp70 are immunodominant proteins synthesized during the *M. tuberculosis* infection. It was shown that these Hsps possess autophosphorylation activity (Preneta et al., 2004). *M. tuberculosis* genome has revealed the presence of heat shock proteins ClpP1, ClpP2, ClpC1, ClpX and ClpC2. The ClpC1 of *M. tuberculosis* has been shown to have an inherent ATPase activity, and to prevent protein aggregation as a chaperone in the absence of any adaptor protein (Kar et al., 2008). *M. tuberculosis* ClpC1 has also been shown to interact with ClpP2 (Singh et al., 2006). *M. tuberculosis* ClpC1 has been shown to interact with ResA, an anti sigma factor which is degraded by ClpC1P2 protease *in vitro* (Barik et al., 2010). Knockdown of ClpC1 in *M. smegmatis* and *M. tuberculosis* showed inhibition of RseA degradation indicating a regulatory role of Clp proteins in *M. tuberculosis* (Barik et al., 2010). ClpX is predicted to be essential for *in vivo* survival and pathogenicity and is conserved in *M. tuberculosis, M. leprae, M. bovis* and *M. avium paratuberculosis* (Ribeiro-Guimaraes & Pessolani, 2007). ClpX of *M. tuberculosis* was not able to substitute ClpC1 in ClpC1P2 protease complex (Barik et al., 2010). Knockdown of ClpX in *M. smegmatis* did not prevent the degradation of RseA indicating that ClpP2 does not associate with ClpX for its proteolytic activity (Barik et al., 2010). Leprosy and tuberculosis patients with active disease had shown the presence of antibodies recognizing ClpC in dot ELISA (Misra et al., 1996). The expression of ClpX was found to be upregulated in *M. tuberculosis* upon macrophage infection (Dziedzic et al., 2010). FtsZ is a protein known to assemble at the midcell division site in the form of a Z-ring. It is crucial for initiation of the cell division process in eubacteria. ClpX has been shown to interact with FtsZ in *M. tuberculosis* (Dziedzic et al., 2010). The crystal structure of *M. tuberculosis* caseinolytic protease, ClpP1 showed a disordered conformation of the residues in the catalytic triad, which makes the protein inactive (Ingvarsson et al., 2007). ClpP of *M. tuberculosis* has been studied as a target for drug designing (Tiwari et al., 2010). *M. avium*, the causative agent of paratuberculosis (Johne's disease) and an economic problem for beef, dairy and sheep industries showed increased expression of ClpB gene during infection (Hughes et al., 2007). ClpB, of *M. bovis* BCG showed reactivity with sera of TB patients suggesting it to be an antigen target of the human immune response to mycobacteria (Bona et al., 1997). There are specific transcription factors that are involved in the regulation as well as transcription of Hsps during different conditions. ClgR, a clp gene regulator of *M. tuberculosis* activates the transcription of at least ten genes, including four that encode protease systems ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c, and three that encode chaperones Acr2, ClpB and the chaperonin Rv3269 (Estorninho et al., 2010). This transcriptional activation and regulatory function of ClgR is very important in the replication of bacteria inside the macrophages. It has been shown that ClgR deficient *M. tuberculosis* is not able to resist the pH inside macrophage post infection (Estorninho et al., 2010). The mechanism by which mycobacteria return to a replicating state after a nonreplicating state, when exposed to low oxygen tension conditions is not clearly understood. ClgR is also implicated in the resumption of replicating state after hypoxia in *M. tuberculosis* (Sherrid et al., 2010).
