**3. Heat shock proteins**

Heat shock proteins (Hsps) are well conserved and universal in all organisms. The expression of Hsps is highly increased under stress conditions such as hypoxia, nutrient starvation and oxygen radical (Richter et al., 2010; Tyedmers et al., 2010). Heat shock and other types of stresses lead to protein aggregation and unfolding of proteins. However, the most deleterious effect is the collapse of intermediary filament, tubulin and actin networks which leads to complete loss of localization and breakdown of intracellular transport and fragmentation of ER (Toivola et al., 2010). Hsps have cytoprotective roles, and under the stress conditions they maintain the cellular organization and homeostasis. Hsps are expressed at significant level in all eukaryotic and prokaryotic cells under normal conditions at physiological temperature. There is a high level of conservation of Hsps indicating a fundamental role played by these proteins in cellular processes (Moseley, 1997; Richter et al., 2010). Hsps were initially discovered in *Drosophila melanogaster* larvae as chromosomal puffs when it was exposed to heat shock (Tissieres et al., 1974). Subsequently, several Hsps were discovered in the following years. Many heat shock proteins work as molecular chaperones that are essential for maintaining cellular functions by preventing misfolding and aggregation of nascent polypeptides and by facilitating protein folding of conformationally altered proteins (Lanneau et al., 2010; Tyedmers et al., 2010).

*tuberculosis* survival (Armstrong & Hart, 1975). After phagocytosis and replication of pathogenic bacteria within macrophages, the infected cells migrate into tissues where additional immune cells are recruited to form a granuloma which consists of T cells and *M. tuberculosis*-infected macrophages (Grosset, 2003). The granuloma subsequently develops central areas of necrosis called caseum. This mass of cells of immune system and the bacteria are all dead cells. The surviving bacilli exist in a latent state and can become reactivated to develop active disease (Grosset, 2003). The latent infection in the asymptomatic individuals serves as a large reservoir of the bacterium. The biology of the latent state of the bacterium is not completely understood, however it is accepted that the latent state bacilli are

Inside the macrophages, *M. tuberculosis* encounters many stress conditions like nitric oxide generated by inducible nitric-oxide synthase, nutrient starvation or carbon limitated condition, and reactive oxygen species (ROS) by the phagosomal NADPH oxidase (Farhana, 2010; Ehrt, 2009; Butler, 2010; Beste, 2007; Axelrod, 2008). A large number of studies have been undertaken to understand the survival of *M. tuberculosis* under stress such as heat, reduced oxygen or hypoxia, nutrient starvation, reactive nitrogen intermediates (RNI), antimicrobial molecules and downshift in pH (Chan et al., 1992; Farhana et al., 2010; Firmani & Riley, 2002; Lowrie, 1983; Wayne & Sohaskey, 2001). It has been suggested that the bacteria enter the non-growth or stationary phase during such stress conditions (Wayne & Sohaskey, 2001). *M. tuberculosis* also survives the lethal effects of RNI and antimicrobial molecules produced by activated macrophages and other cell types (Chan et al., 1992). The intracellular pathogen has the ability to survive inside the host macrophage in spite of the microbicidal effector functions of the macrophages. The bacterium responds to the stress conditions by genome wide changes in gene expression including the induction of a transient expression of a well conserved set of genes encoding heat shock or heat stress

Heat shock proteins (Hsps) are well conserved and universal in all organisms. The expression of Hsps is highly increased under stress conditions such as hypoxia, nutrient starvation and oxygen radical (Richter et al., 2010; Tyedmers et al., 2010). Heat shock and other types of stresses lead to protein aggregation and unfolding of proteins. However, the most deleterious effect is the collapse of intermediary filament, tubulin and actin networks which leads to complete loss of localization and breakdown of intracellular transport and fragmentation of ER (Toivola et al., 2010). Hsps have cytoprotective roles, and under the stress conditions they maintain the cellular organization and homeostasis. Hsps are expressed at significant level in all eukaryotic and prokaryotic cells under normal conditions at physiological temperature. There is a high level of conservation of Hsps indicating a fundamental role played by these proteins in cellular processes (Moseley, 1997; Richter et al., 2010). Hsps were initially discovered in *Drosophila melanogaster* larvae as chromosomal puffs when it was exposed to heat shock (Tissieres et al., 1974). Subsequently, several Hsps were discovered in the following years. Many heat shock proteins work as molecular chaperones that are essential for maintaining cellular functions by preventing misfolding and aggregation of nascent polypeptides and by facilitating protein folding of conformationally

metabolically less active (Wayne & Sohaskey, 2001).

altered proteins (Lanneau et al., 2010; Tyedmers et al., 2010).

proteins.

**3. Heat shock proteins** 

The predominant class of Hsps is of molecular chaperones (Ellis & Hemmingsen, 1989). The molecular chaperones are further grouped into five major families based upon their molecular masses. These families are Hsp100, Hsp90, Hsp70, Hsp60 and small heat shock protein (sHsps) (Richter et al., 2010). The classification is based on their related functions and sizes, using the conventional nomenclature adopted after the Cold Spring Harbor Meeting of 1996 (Hightower & Hendershot, 1997). The molecular chaperones not only facilitate the proper folding of proteins but many times direct improperly folded proteins for destruction. In recent years, multiple chaperone-assisted degradation pathways have emerged, in which chaperones associate with a protease present inside the cell to degrade a misfolded protein (Gottesman, 2003, Kettern et al., 2010). Several other small heat inducible molecular chaperones, like Hsp33 are also known (Jakob et al., 1999).

The Hsp100 family consists of a group of ATPases associated with cellular activities (AAA+) family of ATP-dependent chaperones that transfers aggregated protein into a proteolytic chamber of an associated protease. These energy-dependent proteases, also known as caseinolytic proteases, Clp or Ti, are involved in a number of cellular activities, such as the degradation of proteins misfolded as a result of various types of stresses, the regulation of short-lived proteins and the housekeeping removal of dysfunctional proteins, which include denatured and aggregated polypeptides (Gottesman et al., 1997a, Gottesman et al., 1997b). The members of Hsp100 family include ClpA, ClpB, ClpC, ClpE, ClpX, ClpY and others (Kirstein et al., 2009). Hsp100 proteins have either one or two copies of a conserved ATPase, AAA+ core domain. Hsp100 family is further divided into two subclasses. The class I family members that include ClpA-E and L, contain two ATPase domains. The class II family members contain one ATPase domain, and include ClpX and ClpY (Lindquist & Craig, 1988; Schirmer et al., 1996). The Clp proteins form hexameric structure with one nucleotide binding site in each monomer of Class II and two nucleotide binding sites in Class I (Schirmer et al., 1996). These ATP-dependent chaperones associate with a protease, ClpP or ClpQ forming an oligomeric enzyme which assembles into ring-like or barrel like structure, containing a cavity within the centre of the macromolecular structure (Gottesman, 2003). The central cavity is also known as the proteolytic chamber, where unfolded protein substrates are translocated and subsequently degraded by the proteolytic site (Gottesman, 2003). Degradation of structured protein substrates requires the presence of ATP (Baker & Sauer, 2006). Unlike the other class I Clp proteins, ClpB does not associate with any protease to direct substrates for degradation (Lee et al., 2004).

Hsp90 is present mostly in cytosol of bacteria and eukaryotes, and is upregulated under stress (Welch & Feramisco., 1982). This chaperone is different in a way that it is not very promiscuous in substrate binding as it does not bind unfolded proteins rather it binds to native like proteins (Jakob et al., 1995). Under stress conditions two of the Hsp90 family proteins, namely yeast Sti1 and the propyl isomerise, Cpr6 are upregulated (Pearl & Prodromou., 2006).

Hsp70 family consists of highly conserved chaperones. All Hsp70 proteins bind ATP and under physiological conditions prevent the aggregation of proteins, and also refold aggregated proteins (Kiang & Tsokos, 1998). The activity of Hsp70 is regulated by co-factors. Much of the functional diversity of Hsp70s is driven by a diverse class of cofactors named J proteins or Hsp40 (Kampinga & Craig, 2010). The major members of the Hsp70 family include HSC 70 (heat shock cognate 70), mitochondrial GRP 75 and GRP 78 (Shi & Thomas,

Heat Shock Proteins in *Mycobacterium tuberculosis*:

increased under heat shock (Patel et al., 1991).

(Gahan & Hill, 1999).

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

Involvement in Survival and Virulence of the Pathogen 261

virulence and survival of *L. monocytogenes* in macrophages (Rouquette et al., 1998). In *Salmonella typhimurium* the Clp protease, ClpP is involved in maintaining the level of Sigma factors inside the bacterium; disruption of ClpP leads to decreased virulence in mice (Webb et al., 1999). ClpP mutation significantly attenuated the virulence of *Streptococcus pneumoniae*  in murine intraperitoneal infection model (Kwon et al., 2003). Disruption of the genes for ClpXP protease in *Salmonella enterica* serovar typhimurium results in loss of virulence in mice; these mutants were more sensitive to the intracellular environment of the macrophage

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

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

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, 1992).

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 as Hsp60 and Hsp10.

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