Tài liệu Báo cáo khoa học: Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv ppt

REVIEW ARTICLE Survival mechanisms of pathogenic Mycobacterium tuberculosis H 37 Rv Laxman S. Meena and Rajni Institute of Genomics and Integrative Biology, Delhi, India Introduction Five decades of tuberculosis (TB) control programs using potentially efficacious drugs have failed to reduce prevalence of infection by the causative organism, Mycobacterium tuberculosis, in most parts of the world [1]. A large number of individuals (more than three billion) have been vaccinated with Bacillus Calmette- Gue ´ rin (BCG), but TB still kills more than 50 000 people every week and approximately one-third of the world’s population is asymptomatically infected by M. tuberculosis [2]. It is estimated that 200 million people will display symptoms and that 35 million will die of TB between 2000 and 2020 if control and pre- ventive measures are not strengthened (World Health Organization Annual Report, 2000). TB accounts for 32% of the deaths in HIV infected individuals [3]. The situation is exacerbated by the emergence of multi- drug-resistant TB [4] and the catastrophic nexus between AIDS and TB [5,6]. A prerequisite for effec- tive control is an understanding of the host–pathogen interaction and its contribution to the development of diseases. Our knowledge about how M. tuberculosis enters the host cells is currently limited. Mycobacterium tuberculosis has evolved several mechanisms to circumvent the hostile environment of the macrophage, its primary host cell (Figs 1 and 2). Despite extensive research, our knowledge about the virulence factor(s) of M. tuberculosis is quite inade- quate. Understanding the molecular mechanisms of M. tuberculosis pathogenesis will provide insights into the development of target-specific drugs or effective Keywords dormancy; host cell; lysosome; Mycobacterium; phagosome; signaling transduction; tuberculosis; virulence factor Correspondence L. S. Meena, Institute of Genomics and Integrative Biology, Mall Road, Delhi- 110007, India Fax: +91 11 27667471 Tel: +91 11 27666156 E-mail: meena@igib.res.in; laxmansm72@yahoo.com (Received 2 February 2010, revised 12 March 2010, accepted 29 March 2010) doi:10.1111/j.1742-4658.2010.07666.x Mycobacterium tuberculosis H 37 Rv is a highly successful pathogen and its success fully relies on its ability to utilize macrophages for its replication and, more importantly, the macrophage should remain viable to host the Mycobacterium. Despite the fact that these phagocytes are usually very effective in internalizing and clearing most of the bacteria, M. tuberculosis H 37 Rv has evolved a number of very effective survival strategies, including: (a) the inhibition of phagosome–lysosome fusion; (b) the inhibition of phagosome acidification; (c) the recruitment and retention of tryptophan- aspartate containing coat protein on phagosomes to prevent their delivery to lysosomes; and (d) the expression of members of the host-induced repeti- tive glycine-rich protein family of proteins. However, the mechanisms by which M. tuberculosis H 37 Rv enters the host cell, circumvents host defenses and spreads to neighboring cell are not completely understood. Therefore, a better understanding of host–pathogen interaction is essential if the glo- bal tuberculosis pandemic is ever to be controlled. This review addresses some of the pathogenic strategies of the M. tuberculosis H 37 Rv that aids in its survival and pathogenicity. Abbreviations BCG, Bacillus Calmette-Gue ´ rin; LAM, lipoarabinomannan; PE-PGRS, a repetitive glycine-rich protein family; TACO, tryptophan-aspartate containing coat protein; TB, tuberculosis. 2416 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS vaccine candidates for the treatment of the disease. A variety of mechanisms have been suggested to con- tribute towards the survival of Mycobacterium within macrophages. These mechanisms are shown as a sche- matic representation in Fig. 2. The present review aims to summarize the mechanisms that M. tuberculosis uses to establish itself with the phagosomes of the host macrophages, with an emphasis on recent advances in the field of mycobacterial pathogenesis. Survival strategies employed by M. tuberculosis to survive in host cells Cell wall virulence factors When Mycobacteria are stained by Gram staining, they cannot be decolorized by acid alcohol and are there- fore classified as acid-fast bacilli. Acid fastness is largely a result of the high content of mycolic acids, Rough endoplasmic reticulum Golgi appartus Nucleus Mitochondria Vesicles Lipofuscin Engulfed material Phagocytosis Phagosome Lysosome Phagosome lysosome fusion Killing of ingested pathogen Release of digested material by exocytosis Fig. 1. Detailed structure of a macrophage showing a typical process of phagocytosis. Phagosome Lysosome Inhibition of fusion of phagosome harbouring Mycobacteria with lysosome TACO protein on phagosome harbouring mycobacteria TACO Proton ATPase- Pump Virulence Proteins Expression of virulence proteins of PE-PGRS family Inhibition of acidification of phagosome harbouring Mycobacteria Protection from reactive oxidative radicals Fusion H + O 2 – . OH. H 2 O 2 NO. AD BE C Fig. 2. Key factors of the survival mecha- nisms involved in the phagosome matura- tion arrest of Mycobacterium tuberculosis inside macrophages. L. S. Meena and Rajni Survival strategies of mycobacteria in host FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2417 long chain cross-linked fatty acids and other cell-wall lipids in the cell wall [7]. Mycolic acid and other lipids are linked to underlying arabinogalactan and peptido- glycan [8]. A variety of unique lipids, such as lipoara- binomannan (LAM), trehalose dimycolate and phthiocerol dimycocerate, anchor noncovalently with the cell membrane and appear to play an important role in the virulence of M. tuberculosis [9]. Lipids such as cord factor (surface glycolipid that is toxic to mam- malian cells and is also an inhibitor of polymorphonu- clear leukocyte migration) induce cytokine- mediated events [10,11], which may also contribute to virulence. Treatment of Mycobacterium avium by isoni- azid disrupts mycolic acid biosynthesis, which is responsible for the cording (serpentine cording) phenomenon, and thereby renders the mycobacteria hydrophobic [12]. In the case of Mycobacterium smegmatis, enhanced permeability as a result of disrup- tion of a mycolate ⁄ cording factor gene causes reduced growth both in vitro and in vivo [13]. Disruption of the gene involved in mycolic acid cyclopropanation was shown to alter cording properties and reduce virulence [14]. Using whole genome transpositional mutagenesis techniques, 30 mutants of M. tuberculosis were selected from a total screen of approximately 2500 mutants that showed reduced growth. Seven of these mutants had insertion within a locus involved in the synthesis of phthiocerol dimycocerosate, an abundant compo- nent of cell wall biosynthesis [15]. Phthiocerol dimyco- cerosate was subsequently shown to help entry of Mycobacterium leprae into peripheral nerve cells by binding to nerve cell laminin protein [16]. The majority of exported proteins and protective antigens of M. tuberculosis are a triad of related gene products called the antigen 85 complex, each having fibronectin binding capacity and thus an important role in disease pathogenesis [17]. LAM is also a major constituent of mycobacterial cell wall and has been shown to induce tumor necrosis factor release from the macrophages [18], which plays a prominent role in bacterial killing. Studies have shown that LAM acts at several levels and that it can scavenge potentially cytotoxic oxygen free radicals, inhibit protein kinase C activity and block the tran- scriptional activation of gamma interferon inducible genes in human macrophages such as cell lines, and hence contribute to the persistence of mycobacteria within mononuclear phagocytes [19]. Host cell surface receptors M. tuberculosis appears to gain entry into macrophages via cell surface molecules, including those of the inte- grin family CR1 and CR3 complement receptors [20] and the mannose receptors [21]. By contrast, M. avium enters macrophages via avb3, another receptor of inte- grin family [22]. Unlike other bacteria, pathogenic mycobacteria are opsonized with C3 peptides in an entirely different way, involving the recruitment of the complement fragment C2a to form a C3 convertase and the generation of opsonically active C3b in the absence of early activation components [23]. Individual strains of M. tuberculosis can vary in their modes of interaction with CR3, by interacting with distinct domains of the receptor [24]. It has been shown that mannose receptors bind the virulent Erdman and H 37 Rv strains but not the avirulent M. tuberculosis H 37 Ra strain. This difference in binding may arise because strain H 37 Rv has ligands, such as LAM, that bind to mannose receptors at different sites compared to the M. tuberculosis H 37 Ra strain [25]. Furthermore, it has been suggested that Fc receptor-mediated intake of mycobacteria may involve distinct intracellular traf- ficking for the virulent M. tuberculosis [26]. The relative contribution of various macrophage receptors, such as complement receptors CR1, CR3 and CR4, mannose receptor, lung surfactant protein receptors, CD14, scavenger receptors and Fc receptors, in the intracellular fate and survival of M. tuberculosis is still far from being understood [24]. Successful pathogens (e.g. Salmonella typhi) appear to survive in phagosomes by entering a receptor-mediated pathway that is not coupled to the activation of macrophage antimicrobial mechanisms, such as the production of reactive oxygen or nitrogen intermediates [27]. How- ever, to date, it is not yet clear how mycobacteria use the advantage of selective receptor-mediated intracellu- lar survival as a pathogenic strategy. It is possible that the distinct routes of entry of M. tuberculosis result in different cytokine secretion responses or different downstream activation signals in the host macrophages, leading to the differential survival of this pathogenic bacteria. Inhibition of phagosome–lysosome fusion Both inhibition of growth and killing of intracellular pathogens within the host cell of the mononuclear phagocyte lineage are considered to be dependent on phagosome–lysosome fusion [28]. Immediately after engulfment by macrophages, most tubercle bacilli are directed to phagolysosomes [29]. Subsequently, how- ever, individual M. tuberculosis bud out from the fused phagolysosomes into vacuoles that fail to fuse to the secondary lysosomes and thus escape lysosomal killing. Thus, temporary residence within a phagolysosome Survival strategies of mycobacteria in host L. S. Meena and Rajni 2418 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS stimulates a response to the intracellular environment in M. tuberculosis that facilitates its long-term survival and reproduction. Sulfatides (anionic trehalose glycolipids) of M. tuberculosis also have an antifusion effect [30]. M. tuberculosis can produce ammonia in abundance, which is considered to be responsible for the inhibitory effect of the culture supernatant of virulent mycobacte- ria on phagolysosomal fusion [31]. Ammonium chloride affects the movement of lysosomes by alkalizing the in- tralysosomal compartment [32] and, as a result, it diminishes the potency of intralysosomal enzymes via alkalization. Live M. tuberculosis were shown to infect human macrophages in the presence of low cytosolic Ca 2+ , which is correlated with inhibition of phago- some–lysosome fusion and intracellular viability. It was suggested that defective activation of the Ca 2+ dependent effector proteins calmodulin and calmodulin- dependent protein kinase 2 contributes to the intracellu- lar pathogenesis of tuberculosis [33]. Inhibition of phagosomal acidification The restricted fusogenicity of the mycobacterial vacuole may extend beyond limiting the access of lysosomal hydrolases to the bacilli. It has been reported that vacu- oles containing M. avium are less acidic than neighbor- ing lysosomes [31,34]. Within M. avium, the absence of a vesicular proton-ATPase pump results in a lack of acidification of phagosomes [35]. Recently, a role for natural resistance-associated macrophage protein 1 has been demonstrated [36] in directly promoting H + -ATPase-dependent acidification of Mycobacterium bovis BCG phagosomes in peritoneal macrophages. Maturation of phagosomes M. tuberculosis residing within host phagosomes modi- fies the maturation of the phagosomal compartment and enhances intracellular survival. This maturation leads to the inhibition of phagolysosomal fusion. Moreover, the aberrant expression of Rab5 on the phagosomes containing M. tuberculosis causes the mat- uration arrest of these phagosomes at the early endosomal stage [37]. Phagosomes containing inert particles or avirulent bacteria transiently display Rab5, whereas phagosomes containing virulent M. tuberculosis exhibit a persistent display of Rab5 [37]. Recruitment and retention of tryptophan-aspartate containing coat protein (TACO) on phagosome wall Recruitment and retention of the host protein TACO to phagosomes harboring mycobacteria prevents bacterial delivery to lysosomes [38]. TACO⁄ coronin-1 is an actin binding protein known to associate with cholesterol within the plasma membrane [39]. Reten- tion of TACO on the phagosomal wall allows the mycobacteria to escape the bactericidal action of macrophages [38]. Vitamin D 3 and retinoic acid down- regulate TACO gene transcription in a dose-dependent manner. This down-regulation occurs through the modulation of this gene via the VDR ⁄ RXR response sequence present in the promoter region of TACO gene. Treatment with vitamin D 3 and retinoic acid inhibits mycobacterial entry, as well as survival within macrophages [40]. Moreover, TACO-mediated uptake of mycobacteria depends on cholesterol [39]. Dormancy or persistence within the host macrophages M. tuberculosis has the ability to remain dormant within host cells for years at the same time as retaining the potential to be activated. The dormancy or latency of M. tuberculosis allows the bacterium to escape the activated immune system of the host. Persistence of M. tuberculosis in mice is facilitated by isocitrate lyase, a glyoxylate shunt enzyme that is essential for the metabolism of fatty acids [41]. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. Several genes were identified as being preferentially expressed when Mycobacterium marinum resides in the host granulomas and ⁄ or macrophages [42]. Two of the genes were found to be homologs of genes for M. tuberculosis PE ⁄ PE-PGRS, a family encoding numerous repetitive glycine-rich proteins of unknown function(s). The mutation of these two genes for PE-PGRS produced M. marinum strains that were incapable of replication in macrophages. The strains exhibited decreased persistence in granulomas, thereby suggesting a direct role for PE-PGRS proteins in mycobacterial virulence. Hypoxia was also observed to be a major factor in inducing the nonreplicating persis- tence of tubercle bacilli [43]. Protection against oxidative radicals The macrophages offer a hostile environment to intra- cellular bacteria by producing a vast array of chemi- cals such as reactive oxygen and nitrogen radicals. However, the virulent Erdman strain of M. tuberculosis overexpresses a protein that cyclopropanates mycolic acid double bonds, resulting in a ten-fold lower suscep- tibility to peroxide [44]. Also, the oxyR (i.e. a sensor L. S. Meena and Rajni Survival strategies of mycobacteria in host FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2419 of oxidative stress and a transcriptional activator that induces the expression of detoxifying enzymes such as catalase ⁄ hydroperoxidase) of M. tuberculosis has numerous deletions and frameshift mutations giving the appearance of a pseudogene [45]. Perhaps the pro- tection afforded by cyclopropanated cell wall compo- nents has rendered oxyR superfluous in pathogenic mycobacteria. Superoxide dismutases play an impor- tant role in protection against oxidative stress and so contribute to the pathogenicity of many bacterial species [46]. Virulence genes of M. tuberculosis Initial efforts aimed at identifying the genes involved in the pathogenesis of M. tuberculosis involved the cloning and expression of random genomic DNA frag- ments of pathogenic bacteria into surrogate hosts such as Escherichia coli, followed by the analysis of survival of recombinant E. coli in macrophage cell lines. To identify the genes involved in the invasion of macro- phages by M. tuberculosis, a gene fragment mce was identified that encodes a 52 kDa protein conferring E. coli with the ability to invade HeLa cells and sur- vive within the host macrophages [47]. The intracellu- lar survival of bacteria was impaired with the spontaneous loss of DNA from the transformants. Four copies of the mce gene have been identified in the M. tuberculosis genome and have been designated as mce1, mce2, mce3 and mce4 [48]. The exact function of Mce1 is still unknown; it appears to serve as an effec- tor molecule expressed on the surface of M. tuberculo- sis that is capable of eliciting plasma membrane perturbations in nonphagocytic mammalian cells [49]. In another study, the gene encoding Mce3 protein was disrupted in the vaccine strain M. bovis BCG [50]. The mutant strain exhibited a reduced ability to invade nonphagocytic HeLa cell lines compared to the wild- type BCG, supporting the idea that this gene is involved in the invasion host tissues. M. smegmatis has been used as a surrogate host for cloning, expressing genes and constructing genomic libraries of M. tuberculosis [51,52]. To identify the genes essential for survival of mycobacteria within macrophages, a plasmid library was constructed by using genomic DNA from M. tuberculosis and electro- porated into M. smegmatis [53]. The transformants were used to infect the human macrophages cell line U-937, and one transformant (eis) was isolated that showed an enhanced survival over a period of 48 h compared to the wild-type M. smegmatis [53]. The eis gene, which encodes a 42 kDa protein, confers M. smegmatis with the ability to resist killing by host macrophages. The function of the Eis protein is still unknown. It has been suggested that the secreted pro- teins of mycobacteria have a profound influence on its pathogenicity. It was found that the disruption of an erp gene of M. tuberculosis encoding a secretory pro- tein effects the survival of M. tuberculosis in host mac- rophages [54]. In many Gram-negative bacteria, iron-regulated genes are essential for the expression of full virulence [55]. It is likely that the acquisition of iron by M. tuber- culosis is also essential for growth and survival during the course of infection. M. tuberculosis synthesizes two distinct iron-regulated siderophores: the cell surface- associated mycobactin and the excreted siderophore, exochelin [56]. The mbtB gene, which is involved in the biosynthesis of siderophores, was disrupted in M. tuberculosis and the resulting mutant was observed to have a restricted growth in iron-depleted conditions [57]. The mutant also exhibited stunted growth pattern in human monocyte cell line THP-1, suggesting a role for siderophores in virulence. M. tuberculosis and other mycobacterial species also produce a number of iron-regulated membrane pro- teins [56]. For example, iron-dependent regulatory pro- tein (IdeR) of M. tuberculosis has been characterized as a functional homolog of the diphtheria toxin repres- sor from Corynebacterium diphtheriae [58,59]. The ideR gene was shown to be necessary for high-level expres- sion of the SodA and INH proteins that are involved in the pathogenesis of mycobacteria [60]. It was revealed that the anti-apoptosis activity was a result of the type-1 NADH- dehydrogenase of M. tuberculosis and the main subunit of this multicom- ponent complex is encoded by the gene for Nuo G. Deletion of nuo G in M. tuberculosis resulted in its inability to inhibit macrophage apoptosis and signifi- cantly reduced its virulence [61]. Another gene, named fad D33, encoding an acyl-coenzyme A synthase, plays an important role in M. tuberculosis virulence by sup- porting growth in the liver [62]. Several other genes demonstrated to be essential for the survival of myco- bacteria in macrophages are shown in Table 1. Modulating host signal network A new perspective in the pathogenesis of M. tuberculo- sis is the exploitation of host cell signaling pathways by the pathogen. Upon infection, the phosphatases and kinases of several pathogenic bacteria modify host proteins and help in the establishment of the disease. The uptake of M. tuberculosis by macrophages is associated with a number of early signaling events, such as the recruitment and activation of members of Survival strategies of mycobacteria in host L. S. Meena and Rajni 2420 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS the Src family of protein tyrosine kinases. These kinas- es result in the increased tyrosine phosphorylation of multiple macrophage proteins and the activation of phospholipase D [82]. Activation of protein tyrosine kinases appears to enhance stimulation of phospholi- pase D activity and the associated increase in phospha- tidic acid. Phosphatidic acid may trigger a number of downstream processes that are necessary for membrane remodeling during phagocytosis and the intracellular survival of M. smegmatis in host cells [83]. Further- more, LAM from the virulent species of M. tuberculo- sis possesses the ability to modulate signaling pathways linked to bacterial survival by phosphoryla- tion of an apoptotic protein in the phosphatidylinositol 3-kinase-dependent pathway in THP-1 cells [84]. Many regulatory proteins or enzymes commonly known as G-proteins play a vital role in cell signaling by binding and hydrolyzing GTP to GDP [85]. Despite their common biochemical function of GTP hydrolysis, these proteins are associated with diverse biological functions. In eukaryotes, G-proteins are classified into three main groups: Ras and its homologs; the transla- tion elongation factors [86], Tu and G; and the a subunits of heterotrimeric G-proteins. All members of this group share a common structural core, suggesting a common evolutionary origin for these proteins. The members of G-protein superfamily are known to play a complex array of functions in eukaryotes, such as, hor- mone action, visual transduction and protein synthesis. By contrast to the eukaryotic counterparts, the function of most of the universally conserved bacterial GTPases is still poorly understood. In recent years, there have been significant advances in the research related to the GTP-binding protein in the prokaryotes. Table 1. Genes involved in virulence of mycobacteria. Serial number Gene name Gene number Function References 1 aceA Rv0467 Isocitrate lyase ⁄ dormancy [63] 2 mceD Rv0170 Cell invasion [64] 3 cmaA, mmaA4 Rv3392c ⁄ Rv0503c ⁄ Rv0642c Mycolic acid biosynthesis [65] 4 sigE ⁄ sigH Rv1221 ⁄ Rv3223c Sigma factors [66] 5 Acr Rv2031c Growth in macrophages [67] 6 drrC Rv2938 ABC transporter [68] 7 – Rv3718c PE-PGRS family [42] 8 erp Rv3810 Cell-wall associated surface proteins [54] 9 ideR Rv2711 Iron-dependent repressor [69] 10 glnA Rv2220 Nitrogen metabolism [70] 11 aphC Rv2428 Oxidative stress defense [71] 12 KatG Rv1908c Catalase ⁄ peroxidase [72] 13 fadD26 Rv2930 Lipid metabolism [73] 14 fadD28 Rv2941 Mycocerosis acid synthesis [74] 15 fbpa Rv3804c Mycolyl transferase [75] 16 PKnG Rv0410c Phosphorylates the peptide substrate myelin basic protein at serine residues ⁄ serine ⁄ threonine-protein kinase protein kinase G [76] 17 Pks2 Rv3825c Polyketide synthase PKS2 [77] 18 fadE28 Rv3544c Acyl-coenzyme A dehydrogenase [78] 19 nuoG Rv3151 NADH dehydrogenase I (chain G) NADH-ubiquinone oxidoreductase chain G [61] 20 phoP Rv0757 Positive regulator for the phosphate regulon, required for intracellular growth [79] 21 plcA Rv2351c Phospholipase c 1 plca (mtp40 antigen) [80] 22 plcB Rv2350c Membrane-associated phospholipase c 2 plcb [80] 23 plcC Rv2349c Intracellular survival, by the alteration of cell signaling events or by direct cytotoxicity ⁄ phospholipase c 3 plcc [80] 24 plcD Rv1755c Intracellular survival, by the alteration of cell signaling events or by direct cytotoxicity ⁄ phospholipase c 4 (fragment) plcd. [80] 25 mmpL8 Rv3823c Considered to be involved in the transport of lipids and shown to be required in the production of a sulfated glycolipid, sulfolipid-1 [81] L. S. Meena and Rajni Survival strategies of mycobacteria in host FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2421 Recent studies have shown that bacterial GTPases con- trol vast arrays of function, such as the regulation of ribosomal function and the cell cycle, the modulation of DNA partitioning and DNA segregation [87]. The best known prokaryotic small GTP-binding protein is Era (named for ‘E. coli Ras-like protein’). Era is essen- tial for the growth of E. coli, Salmonella typhimurium and Streptococcus mutans because mutants of Era reveal pleiotropic phenotypes, including alterations in the regulation of carbon metabolism, the stringent response and cell division [88–92]. In E. coli, depletion of Era at 27 °C was shown to cause cell filamentation [92] and a mutation in the GTP-binding domain sup- presses temperature-sensitive chromosome partitioning mutations, indicating that Era is a cell-cycle check- point regulator [89,90]. Interestingly, similar to Era, homologs of Obg (a new subfamily of small GTP-binding protein) are also present both in prokaryotes and eukaryotes [93]. Several bacterial homologs of the Obg subfamily have been characterized, and examples include Obg proteins from Bacillus subtilis, Streptomyces griseus, Streptomy- ces coelicolor, CgtA (CgtA is also called ObgE) proteins from Caulobacter crescentus, E. coli, Vibrio harveyi and YhbZ from Haemophilus influenzae [93–99]. Obg proteins of B. subtilis and Streptomyces species are essential for vegetative growth and the initiation of sporulation [93,94,96,100,101]. The Obg homologue (CgtA) in C. crescentus was shown to be indispensable for growth [97]. Similarly, E. coli homo- log YhbZ (renamed ObgE) has also been reported to comprise an essential gene involved in the chromosome partitioning [95]. Besides these roles for Era and Obg, this category of protein has also been shown to be necessary for the stress-dependent activation of transcription factors. Homologs of the Ras family of GTP-binding proteins have also been shown to contribute to morphology and virulence in several pathogenic fungi [101]. LepA is another member of GTP-binding protein family; however, its exact function is still not clear. Helicobacter pylori resides in the gastric mucus layer, where the pH is in the range 4.5–5.0; therefore, to per- sist in the hostile acidic environment of the stomach, it must survive acid shock and grow at acidic pH. Inacti- vation of an ortholog of the E. coli LepA in H. pylori resulted in the inability of mutant to grow at pH 4.8, suggesting that LepA is essential for the growth of H. pylori under acidic conditions and that it might play a critical role in infection by this pathogen [103]. Microbial pathogens such as mycobacteria have sus- tained a long lasting association with their host because they have evolved sophisticated mechanisms to interfere with the macrophage signaling process and eventually affect the overall phagocytosis process. Keeping in view the importance of G-proteins, one approach to help understand this mechanism would involve looking for the presence of such G-proteins in M. tuberculosis, which might interfere with the cell sig- naling and might be specifically expressed under growth of bacteria in macrophage. Keeping in mind the importance of members of G-proteins in diverse functions such as bacterial growth, survival, stress management and virulence, we investigated the complete genome sequence of M. tuberculosis, aiming to identify the presence of genes encoding the GTP-binding proteins. The genome sequence of M. tuberculosis demonstrated that, in addi- tion to homologs of Obg and Era, the additional family member LepA is also present. In our earlier study, three G-proteins, Era, Obg and LepA of M. tuberculosis, were cloned and expressed in E. coli. Purified proteins showed GTP-binding and hydrolyzing activities [104]. A point mutation in the conserved GTP-binding motif, AspXXGly (Asp to Ala), in Era (Asp-258) and Obg (Asp-212) proteins resulted in the loss of the associated activities, confirming that known key residues in well-established G-proteins are also conserved in mycobacterial homologs. This study confirms that M. tuberculosis harbors functional Era, Obg and LepA proteins. Mycobacterium tuberculosis is an intracellular pathogen and has evolved strategies to survive in the acidic environment of macrophages. Therefore, it would be interesting to determine whether functional Era, Obg and LepA proteins of M. tuberculo- sis, similar to their counterparts in other bacteria, play a crucial role in its survival ⁄ pathogenesis. Protein kinases have been found to coordinate the stress response, the developmental process and patho- genicity in several microorganisms [105]. The presence of functional Ser ⁄ Thr kinases [106] in mycobacteria was reported prior to the release of the complete gen- ome sequence of M. tuberculosis, and the genomic sequence then suggested the presence of eleven putative protein kinases [48]. The serine ⁄ threonine kinases of M. tuberculosis are likely to mediate specific signal transduction events with host pathways. Protein kinas- es G and F may comprise key molecules that change the phosphorylation pattern of host proteins upon infection, thereby promoting bacterial survival [107]. Inhibitors of protein kinases have also been shown to prevent the uptake of M. leprae by peritoneal macro- phages of mice [108]. This suggests that the protein kinases of M. tuberculosis may be involved in modify- ing the host phosphorylation pattern to promote their establishment and survival within the host cells. Survival strategies of mycobacteria in host L. S. Meena and Rajni 2422 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS A major anti-phosphotyrosine reactive protein is present only in strains belonging to M. tuberculosis complex [109]. Thus, protein phosphorylation may play an important role in the pathogenesis of myco- bacteria. It has been shown that M. tuberculosis has two functional tyrosine phosphatases that are secreted into the culture supernatant, and that they may inter- fere within the host cells [110]. Recently, a new transporter family (mmpL) was shown to transport lipid molecules into host cells [9], where they may interact with specific host cellular tar- gets and serve to modulate the host-signaling network. Mycobacterial lipids can be found in host cytoplasm without a mycobacterial presence within the host cells [111]. Stress-induced p38 mitogen-activated protein kinase is a component of M. tuberculosis phagosome arrest. The uptake of Mycobacterium stimulates p38 phosphorylation in the macrophage. EEA1 (i.e. early endosomal autoantigen) plays an essential role in phagosome maturation. EEA1 is recruited to mem- brane by Rab 5 and by PI3P [112]. It was proposed that the PKnH kinase of M. tuberculosis mediates a host signal and triggers events that are responsible for the intracellular survival of the bacterium, thus leading to chronic infection [113]. Conclusions M. tuberculosis, the causative agent of tuberculosis is still a major burden to human health. M. tuberculosis is very unusual among the bacterial pathogens with respect to its ability to persist in the face of host immune responses. The ability of M. tuberculosis to persist within macrophages is well known, although the molecular mechanisms behind this resistance have not been resolved so far. However, the release of the complete genome sequence of M. tuberculosis, as well as recent advances in functional genomics tools (e.g. microarrays and proteomics), in combination with modern approaches, has facilitated a more rational and directional approach towards the understanding of these mechanisms. Therefore, it is clear that a better understanding of these mechanisms and the host–path- ogen interaction will be essential not only to control this pandemic, but also to elucidate the novel features of macrophage defenses and host immune responses. The success of M. tuberculosis during the parasitization of macrophages involves a modulation of the normal progression of the phagosome into an acidic and hydrolytically active phagolysosome, and also avoids the development of localized, productive immune responses against M. tuberculosis in the host. Acknowledgements We thank Rajesh S. Gokhale for making this work possible. We also thank Hemant Khanna (University of Michigan, Flint, MI, USA) for providing valuable suggestions. The authors acknowledge financial sup- port from GAP0050 of the Department of Science and Technology and Council of Scientific & Industrial Research. References 1 Scheindlin S (2006) The fight against tuberculosis. Molecular interventions 6, 124–130. 2 Snider DE, Raviglione M & Kochi A (1994) Global burden of tuberculosis. In Tuberculosis: Pathogenesis, Protection, and Control (Bloom BR ed), pp 2–11. American Society for Microbiology, Washington, DC. 3 Raviglione MC, Snider DE & Kochi A (1995) Global epidemic of tuberculosis: morbidity and mortality of a world wide epidemic. JAMA 273, 220–226. 4 Culliton BJ (1992) Drug-resistant tuberculosis may bring epidemic. Nature (London) 356, 473. 5 Butler D (2000) New fronts in old war. Nature 406, 670–672. 6 Chaisson RE & Slutkin G (1989) Tuberculosis and human immunodeficiency virus infection. J Infect Dis 159, 96–100. 7 Daffe ´ M & Draper P (1998) The envelope layers of mycobacteria with reference to their pathogenicity. Adv Microb Physiol 39, 131–203. 8 Ratledge C (1982) Nutrition, growth, and metabolism. In The Biology of Mycobacterial (Ratledge C & Stanford J eds). pp 186–212. Academic Press Inc, London. 9 Glickman MS & Jacobs WR (2001) Microbial patho- genesis of Mycobacterium tuberculosis: dawn of a disci- pline. Cell 104, 477–485. 10 Indrigo J, Hunter RL & Actor JK (2003) Cord factor trehalose mediates trafficking events during mycobacte- rial 6, 69-dimycolate (TDM) infection of murine mac- rophages. Microbiology 149, 2049–2059. 11 Devergne O, Emilie D, Peuchmaur M, Crevon MC, DiAgay MF & Galanaud P (1992) Production of cyto- kines in sarcoid lymph nodes: preferential expression of interleukin-1-beta and interferon-gamma genes. Hum Pathol 23, 317–323. 12 Barry CE (2001) Mycobacterium smegmatis: an absurd model for tuberculosis? Trends Microbiol 9, 473–474. 13 Liu J & Nikaido H (1999) A mutant of Mycobacte- rium smegmatis defective in the biosynthesis in mycolic acid biosynthesis accumulates meromycolates. Proc Natl Acad Sci USA 96, 4011–4016. L. S. Meena and Rajni Survival strategies of mycobacteria in host FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2423 14 Glickmann MS (2000) A novel mycolic acid cyclopro- pane synthetase is required for coding, persistence and virulence of Mycobacterium tuberculosis. Mol Cell 5, 717–727. 15 Cox JS, Chen B, McNeil M & Jacobs WR Jr (1999) Complex lipids determines the tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402, 79–83. 16 Ng V, Zanazzi G, Timpl R, Talts JF, Salzer JL, Brennan PJ & Rambukkana A (2000) Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae. Cell 103, 511–524. 17 Belisle JT (1997) Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 276, 1420–1422. 18 Chatterjee D (1992) Lipoarabinomannan. Multigly- cosylated form of the mycobacterial mannosylphos- phatidylinositols. J Biol Chem 267, 6228–6233. 19 Chan J, Fan X, Hunter SW, Brennan PJ & Bloom BR (1991) Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages. Infect Immun 59, 1755–1761. 20 Ahearn JM & Fearon DT (1989) Structure and func- tion of the complement receptors, CR1 (CD35) and CR 2 (CD21). Adv Immunol 46, 183–219. 21 Schlesinger LS (1993) Macrophage phagocytosis of vir- ulent but not attenuated strains of Mycobacte- rium tuberculosis is mediated by mannose receptors in addition to complement receptors. J Immunol 150, 2920–2930. 22 Rao SP, Ogata K & Cantanzaro A (1993) Mycobacte- rium avium –M. intracellulare binds to the integrin receptor v3 on human monocyte and monocyte derived macrophages. Infect Immun 61, 663–670. 23 Schorey JS, Carroll MC & Brown EJ (1997) A macro- phage invasion mechanism of pathogenic mycobacteria. Science 277, 1091–1093. 24 Ernst JD (1998) Macrophages receptors for Mycobacte- rium tuberculosis. Infect Immun 68, 1277–1281. 25 Schlesinger LS, Kaufmann TM, Iyer S, Hull SR & Marchiando LK (1996) Differences in mannose recep- tor mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacte- rium tuberculosis by human macrophages. J Immunol 157, 4558–4575. 26 Armstrong J & DiArcy HP (1971) Response of cul- tured macrophages to Mycobacterium tuberculosis, with observation on fusion of lysosomes with phagosomes. J Exp Med 134, 713–740. 27 Ishibashi Y & Arai T (1990) Roles of the complement receptors type 1 (CR 1) and type 3 (CR 3) on phogocy- tosis and subsequent phagosome lysosome fusion in salmonella infected murine macrophages. FEMS Microbiol Immunol 2, 89–96. 28 Moulder JW (1985) Comparative biology of intracellu- lar parasitism. Microbiol Rev 49, 298–337. 29 McDonough KA, Kress Y & Bloom BR (1993) Patho- genesis of tuberculosis Interaction of Mycobacte- rium tuberculosis with macrophages. Infect Immun 61, 2763–2773. 30 Goren MB (1977) Phagocyte lysosomes: interactions with infectious agents, phagosomes, and experimental perturbations in function. Annu Rev Microbiol 31, 507– 533. 31 Gorden AH, Hart PA & Young MR (1980) Ammonia inhibits phagosomes-lysosome fusion in macrophages. Nature 286, 79–81. 32 Hart D, Young MR, Jordan MW, Perkins WJ & Geisow MJ (1983) Chemical inhibitors of phagosome– lysosome fusion in cultured macrophages also inhibit salutary lysosomal movements. A combined micro- scopic and computer study. J Exp Med 158, 477–492. 33 Malik ZA, Shankar SI & Kusner DJ (2001) Mycobac- terium tuberculosis phagosomes exhibit altered calmod- ulin-dependent signal transduction contribution to inhibition of phagosome–lysosome fusion and intracel- lular survival in human macrophages. J Immunol 166, 3392–3401. 34 Crowle AJ, Dahl R, Ross E & May MH (1991) Evidence that vesicles containing living, virulent, Mycobacterium avium in cultured human macrophages are not acidic. Infect Immun 59, 1823–1831. 35 Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins HL, Fok AK, Allen RD, Gluck SL, Heuser J & Russell DG (1994) Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263, 678–681. 36 Hackam DJ, Roptstein OD, Zhang WJ, Cruenheid SP & Grinstein S (1998) Host resistance to intracellular infection: mutation of natural resistance-associated macrophages protein 1 (Nramp 1) impairs phagosomal acidification. J Exp Med 188, 351–364. 37 Clemens DL, Lee B & Horwitz MA (2000) Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phag- osomal fate. Infect Immun 68, 2671–2684. 38 Ferrari G, Langen H, Naito M & Pieters J (1999) A coat protein on phagosomes involved in the intracellu- lar survival of mycobacteria. Cell 97, 435–447. 39 Gatfield J & Pieters J (2000) Essential role for choles- terol in entry of mycobacteria into macrophages. Sci- ence 288, 1647–1650. 40 Anand PK & Kaul D (2003) Vitamin D 3 -dependent pathway regulates TACO gene transcription. Biochem Biophys Res Commun 310, 876–877. 41 McKinney JD, Bentrup KHZ, Munoz-Elias EJ, Miczak A, Chen B, Chan W, Swenson D, Sacchettinl JC, Jacobs WR & Russel DG (2000) Persistence of Survival strategies of mycobacteria in host L. S. Meena and Rajni 2424 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735–738. 42 Ramakrishnan L, Federspiel NA & Falkow S (2000) Granuloma-specific expression of Mycobacterium viru- lence proteins from the glycine-rich PE-PGRS family. Science 288, 1436–1439. 43 Wayne CD & Sohaskey (2001) Nonreplicating persis- tence of Mycobacterium tuberculosis. Annu Rev Micro- biol 55, 139–163. 44 Yuan Y, Lee RE, Besra GS, Belisle JT & Barry CE (1995) Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 92, 6630–6634. 45 Sherman DR, Sabo PJ, Hickey MJ, Arain TM, Mahatras GG, Yuan Y, Barry CE & Stover CK (1995) Disparate response to oxidative stress in saprophytic and pathogenic mycobacteria. Proc Natl Acad Sci USA 92, 6625–6629. 46 Dussurget O, Stewart G, Neyrolles O, Pescher P, Young D & Marchal G (2001) Role of Mycobacte- rium tuberculosis copper-zinc superoxide dismutase. Infect Immun 69, 529–533. 47 Arruda S, Bomfim G, Knights R, Huima-Byron T & Riley LW (1993) Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science 261, 1454–1457. 48 Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE et al. (1998) Deciphering the biology of Mycobacte- rium tuberculosis from the complete genome sequence. Nature 393, 537–544. 49 Chitale S, Ehrt S, Kawamura I, Fujimura T, Shimono N, Anand N, Lu S, Cohen-Gould L & Riley LW (2001) Recombinant Mycobacterium tuberculosis pro- tein associated with mammalian cell entry. Cell Micro- biol 3, 247–254. 50 Flesselles B, Anand NN, Remani J, Loosmore SM & Klein HM (1999) Disruption of mycobacterial cell entry gene of Mycobacterium bovis BCG results in a mutant that exhibits a reduced invasiveness for epithe- lial cells. FEMS Microbiol Lett 177, 237–242. 51 Wieles B, Ottenhoff TH, Steenwijk TM, Franken KL, de Vries RR & Langermans JA (1997) Increased intra- cellular survival of Mycobacterium smegmatis contain- ing the Mycobacterium leprae thioredoxin-thioredoxin reductase gene. Infect Immun 65, 2537–2541. 52 Garbe T, Harris D, Vordermeier M, Lathigra R, Ivanyi J & Young D (1993) Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation. Infect Immun 61, 260–267. 53 Wei J, Dahl JL, Moulder JW, Roberts EA, OiGarra P, Young DB & Freidmann V (1999) Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J Bacteriol 182, 377–384. 54 Berthet FX, Lagranderie M, Gounon P, Laurent-Win- ter C, Ensergueix D, Chavaror P, Thouron F, Mara- nghi E, Pelicic V, Portnoi D et al. (1998) Attenuation of virulence by disruption of the Mycobacterium tuber- culosis erp gene. Science 282, 759–762. 55 Litwin CM & Calderwood SB (1993) Role of iron in regulation of virulence genes. Clin Microbiol Rev 6, 137–149. 56 Wheeler PR & Ratledge C (1994) Metabolism of Mycobacterium tuberculosis.InTuberculosis: Patho- genesis, Protection and Control (Bloom B ed), pp. 353–385. American Society for Microbiology, Washington, DC. 57 Voss D, Rutter JK, Schroeder BG, Su H, Zhu Y & Barry CE (2000) The salicylate derived mycobactin sid- erophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc Natl Acad Sci USA 97, 1252–1257. 58 Schmitt MP, Predich M, Doukhan L, Smith I & Holmes RK (1995) Characterization of an Iron-depen- dent regulatory protein (IdeR) of Mycobacterium tuber- culosis as a functional homolog of the diphtheria toxin repressor (Dtx R) from Corynebacterium diptheriae. Infect Immun 63, 4284–4289. 59 Manabe YC, Saviola BJ, Sun L, Murphy JR & Bishai WR (1999) Attenuation of virulence in Mycobacte- rium tuberculosis expressing a constitutively active iron repressor. Proc Natl Acad Sci USA 96, 12844– 12848. 60 Dussurgett O, Rodriguez M & Smith I (1998) Protec- tive role of Mycobacterium smegmatis IdeR against reactive oxygen species and isoniazid toxicity. Tuberc Lung Dis 79, 99–106. 61 Velmurugan K, Chen B, Miller JL, Azogue S, Gurses S, Hsu T, Glickman M, Jacobs WR Jr, Porcelli SA & Briken V (2007) Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathog 3, e110. doi:10.1371/journal.ppat. 0030110. 62 Rindi L (2002) Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB ⁄ c mice. Microbiology 148, 3873–3880. 63 Honer ZU, Bentrup K, Miczak A, Swenson DL & Russell DG (1999) Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis. J Bacteriol 181, 7161– 7167. 64 Graham JE & Clark-Curtiss JE (1999) Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA 96 , 11554–11559. L. S. Meena and Rajni Survival strategies of mycobacteria in host FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2425 [...]... Regulation of expression of mas and fadD28, two genes involved in production of Dimycocerosyl Phthiocerol, a virulence factor of Mycobacterium tuberculosis J Bacteriol 184, 6796–6802 75 Armitige LY, Jagannath C, Wanger AR & Norris SJ (2000) Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv: effect on growth in culture and in macrophages Infect Immun 68, 767–778... loci essential for the growth of Helicobacter pylori under acidic conditions J Infect Dis 182, 1566–1569 104 Meena LS, Chopra P, Bedwal RS & Singh Y (2008) Cloning and characterization of GTP-binding proteins of Mycobacterium tuberculosis H37Rv Enzyme Microbial Technol 42, 138–144 105 Av-Gay Y & Everett M (2000) The eukaryotic-like Ser ⁄ Thr protein kinases of Mycobacterium tuberculosis Trends Microbiol... Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis Evidence that this lipid is involved in the cell wall permeability barrier J Biol Chem 276, 19845–19854 69 Manabe YC, Hatem CL, Kesavan AK, Durack J & Murphy JR (2005) Both Corynebacterium diphtheriae DtxR(E175K) and Mycobacterium tuberculosis IdeR(D177K) are dominant positive repressors of IdeR-regulated genes in M tuberculosis. .. associated with phenotypic virulence of Mycobacterium tuberculosis Microb Pathog 45, 12–17 ´ 79 Perez E, Samper S, Bordas Y, Guilhot C, Gicquel B & Martı´ n C (2001) An essential role for phoP in Mycobacterium tuberculosis virulence Mol Microbiol 41, 179–187 80 Talarico S, Durmaz R & Yang Z (2005) Insertion- and deletion-associated genetic diversity of Mycobacterium tuberculosis phospholipase C-encoding... Collins DM (1995) Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis Mol Microbiol 15, 1009–1015 73 Infante E, Aguilar LD, Gicquel B & Pando RH (2005) Immunogenicity & protective efficacy of the Mycobacterium tuberculosis fadD26 mutant Clin Exp Immunol 141, 21–28 74 Sirakova TD, Fitzmaurice AM & Kolattukudy PE (2002) Regulation of expression of mas and fadD28, two genes... protein kinase from Mycobacterium tuberculosis Eur J Biochem 244, 604–612 107 Koul A, Choidas A, Tyagi AK, Drlica K, Singh Y & Ullrich A (2001) Serine ⁄ threonine protein kinases PknF and PknG of Mycobacterium tuberculosis: characterization and localization Microbiology 147, 2307–2314 108 Prabhakaran K, Harris EB & Randhawa B (2000) Rugulation by protein kinase of phagocytosis of Mycobacterium leprae... Clin Microbiol 43, 533–538 81 Domenech P, Reed MB & Barry CE (2005) Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance Infect Immun 73, 3492–3501 82 Kusner DJ, Hall CF & Schlesinger LS (1996) Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages J Exp Med 184, 585–595... Overexpression of the cgtA (yhbZ, obgE) gene, coding for an essential GTP-binding protein, impairs the regulation of chromosomal functions in Escherichia coli Curr Microbiol 45, 440–445 Okamoto S, Itoh M & Ochi K (1997) Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation J Bacteriol 179, 170–179 Survival strategies of mycobacteria... phosphorylation in Mycobacterium tuberculosis FEMS Microbiol Lett 124, 203–208 110 Koul A, Choidas A, Treder M, Tyagi AK, Drlica K, Singh Y & Ullrich A (2000) Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis J Bacteriol 182, 5425–5432 111 Beatty WL, Rhoades ER, Ullrich HJ, Chatterjee D, Heuser JE & Russell DG (2000) Trafficking and release of mycobacterial... 235–247 112 Chua J & Deretic V (2004) Mycobacterium tuberculosis reprograms waves of phosphatidylinositol 3-phosphate on phagosomal organelles J Biol Chem 279, 36982– 36992 113 Papavinasasundaram KG, Chan B, Chung JH, Colston MJ, Davis EO & Av-Gay Y (2005) Deletion of the Mycobacterium tuberculosis pknH gene confers a higher bacillary load during the chronic phase of infection in BALB ⁄ c mice J Bacteriol . REVIEW ARTICLE Survival mechanisms of pathogenic Mycobacterium tuberculosis H 37 Rv Laxman S. Meena and Rajni Institute of Genomics and Integrative. disease. A variety of mechanisms have been suggested to con- tribute towards the survival of Mycobacterium within macrophages. These mechanisms are shown