**Heat Shock Proteins in** *Mycobacterium tuberculosis***: Involvement in Survival and Virulence of the Pathogen**

Divya Bajaj and Janendra K. Batra *National Institute of Immunology Aruna Asaf Ali Marg New Delhi India* 

### **1. Introduction**

256 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

Sorokoumova, G.M., Andreevskaya, S.N., Smirnova, T.G., Petrova, E.E., Zhogina, Yu.A.,

Spector, A.A., John, K., & Fletcher, J.E. (1969). Binding of long-chain fatty acids to bovine serum albumin. *Journal of Lipid Research*, Vol. 10, No. 1, (January 1969), pp. 56-67 Stonehouse, M.J., Cota-Gomez, A., Parker, S.K., Martin, W.E., Hankin, J.A., Murphy, R.C.,

Sung, N., Takayama, K., & Collins, M.T. (2004). Possible association of GroES and antigen 85

Takayama, K., Wang, L., & David, H.L. (1972). Effect of isoniazid on the *in vivo* mycolic acid

Wehrli, W., & Staehelin, M. (1971). Actions of the rifamycins. *Bacteriological Reviews*, Vol. 35,

Yamamoto, K., Muniruzzaman, S., Rajagopalan, M., & Madiraju, M.V. (2002). Modulation of

Zhang, Y., Yang, Y., Woods, A., Cotter, R.J., & Sun, Z. (2001). Resuscitation of dormant

phospholipids. *Biochem J.*, Vol. 363, Pt. 2, (April 2002), pp.305-311

*Russian Journal of Biopharmaceuticals*, Vol. 3, No. 2, pp. 19-27

*Microbiol*, Vol. 70, No. 3, (March 2004), pp. 1688-1697

*Chemother*, Vol. 2, No. 1, (July 1972), pp. 29-35

*Res. Commun*., Vol. 284, No. 2, (June 2001), 542–547

No. 3, (September 1971), pp. 290-309

2009), pp. 550-552

(November 2002), pp. 661-676

the survival both susceptible and resistant strains of *Mycobacterium tuberculosis*.

Kalashnikova, T.Yu., Chernousova, L.N., Selishcheva, A.A., & Shvets V.I. (2009). Effects of liposomes of different lipid composition on in vitro growth of *Mycobacterium tuberculosis* H37Rv. *Bull Exp Biol Med*, Vol. 148, No. 5, (November

Chen, W., Lim, K.B., Hackett, M., Vasil, A.I., & Vasil, M.L. (2002). A novel class of microbial phosphocholine -specific phospholipases C. *Mol Microbiol*, Vol. 46, No. 3,

proteins with heat resistance of *Mycobacterium paratuberculosis*. *Appl. Environmental* 

synthesis, cell growth, and viability of *Mycobacterium tuberculosis*. *Antimicrob Agent* 

*Mycobacterium tuberculosis* DnaA protein-adenine-nucleotide interactions by acidic

*Mycobacterium tuberculosis* by phospholipids or specific peptides. *Biochem. Biophys.* 

Tuberculosis (TB) is an infectious disease of global concern. Worldwide TB kills two million people each year. About 90% of those infected with *Mycobacterium tuberculosis* have asymptomatic, latent TB infection (sometimes called LTBI) (Smith, 2003; Wayne & Sohaskey, 2001). Years after initial infection, the bacilli may resume growth, the outcome of which is active TB. If left untreated, the death rate for active TB cases is more than 50%. Approximately 95% of new cases and 98% of deaths occur in developing nations, where human immunodeficiency virus (HIV) infections are common, this is generally because of the unavailability of proper treatment. The causative agent, *M. tuberculosis* has a cell wall which has a very low permeability for most antibiotics and chemotherapeutic agents. Another critical problem is the development of multi-drug resistant TB (MDR-TB) or extremely drug resistant TB (XDR-TB) (Chiang et al., 2010; Eismont, 2009; WHO report, 2010). Every year in the world, around 440,000 new MDR-tuberculosis cases are found due to bacilli that are resistant to the two main antitubercular drugs, isoniazid and rifampicin. The XDR-TB is a recently developed form. The mortality rate in the case of XDR-TB can go from 50 to 100%. *M. tuberculosis* mutants, resistant to any single drug are naturally present in any large bacterial population, irrespective of exposure to drugs. Despite the availability of effective chemotherapy and the moderately protective vaccine, new anti-TB agents are urgently needed to decrease the global incidence of TB (Cox et al., 2006; Ducati et al., 2006).

#### **2. Mycobacterial infection and survival of pathogen inside the host**

On infection, *M. tuberculosis* resides mainly in the host macrophage, inside an endocytic vacuole called the phagosome. The pathogenic mycobacteria inhibit phagosome-lysosome fusion (Hestvik et al., 2005; Pieters & Gatfield, 2002). Lack of maturation of phagosomes containing pathogenic *M. tuberculosis* within macrophages has been widely recognized as a crucial factor for the persistence of mycobacterial pathogen. Mycobacteria have been shown to remain within phagosomes for a long time after infection by EM analysis (Jordao et al., 2008). It is unclear whether blocking of phagosome–lysosome fusion is essential for *M.* 

Heat Shock Proteins in *Mycobacterium tuberculosis*:

Involvement in Survival and Virulence of the Pathogen 259

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

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

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 &

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,

molecular chaperones, like Hsp33 are also known (Jakob et al., 1999).

to direct substrates for degradation (Lee et al., 2004).

Prodromou., 2006).

*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 metabolically less active (Wayne & Sohaskey, 2001).

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 proteins.
