**6.** *Mycobacterium tuberculosis* **responses to acidic stress**

*M. tuberculosis* encounters acidity in the body in a number of locations including within immune cells, macrophages. When macrophages phagocytose tubercle bacilli, phagosomes of unactivated macrophages are limited in their ability to acidify due to the presence of live *M. tuberculosis*. Bacilli can inhibit phagosomal maturation and also inhibit phagosome lysosome fusion (Armstrong and Hart, 1971; Sturgill-Koszycki et al, 1994; Huynh and Grinstein, 2007). Virulent *M. tuberculosis* can exclude a proton ATPase from the phagosome in non-activated macrophages. Exposure to the cytokine INF-γ can result in increased activation of macro‐ phages and these macrophages that phagocytose live virulent *M. tuberculosis* can lower the intra phagosomal pH (Schaible et al,1998; Via et al, 1998; MacMicking et al, 2003; Ehrt and Schnappinger, 2009). This pH's can be toxic to bacilli either killing them, or inhibiting their growth. The robustness of the response seems to lie in the activation and efficiency of the host's immune response. Anything that interferes with the host's immune status can negatively impact acidic modulation within phagosomes, and lead to more mycobacterial replication. In addition, the tubercle bacillus' ability to respond to acidic stress will likely affect the outcome of the infection.

Mycobacteria seem to bear an intrinsic ability to resist acidic stress. They have a thick waxy cell wall as well as an outer membrane that can resist acidic stress. This physical barrier may serve to inhibit entry of toxic protons, and anything that interferes with this barrier could increase acid susceptibility. Many mutants that are acid susceptible lie in genes that affect cell wall and lipid metabolism (Table 1.). Environmental mycobacteria are found in conditions that may be acidic and can grow at pHs as low as 4.0 (Santos et al, 2007). Pathogenic mycobacteria have evolved to resist acidic stress, and potentially share similar mechanisms with their environmental cousins (Kirschner et al, 1992; Kirschner et al, 1999).

Although *Mycobacterium smegmatis* has been found to have an acid tolerance system it is not known if *M. tuberculosis* also possesses one. However, a large number of genes are upregulated due to acidic stress in *M. tuberculosis*. Interestingly when *M. tuberculosis* is engulfed by the phagosomes of macrophages many genes are upregulated, and when cocanamycinA is added which interferes with the development of acidity, 80% of genes in *M. tuberculosis* that are normally upregulated in the phagosomes fail to do so (Rohde et al; 2007). This is an indication that acidity is one of the main environmental signals *M. tuberculosis* experiences *in vivo*.

A number of genes that are upregulated by acidic stress have been identified in previous studies. Looking at rapid response to acidity at 15 or 30 minutes it was found that genes involved in cell wall ultrastructure were induced (Fisher et al, 2002). The *mymA* operon was induced in this study, and is under the control of VirS which is an AraC/XylS family tran‐ scription factor (Singh et al, 2005). The *lipF* promoter of *M. tuberculosis* is upregulated, but requires a longer time frame (Saviola et al, 2001). It fails to be upregulated at 30 minutes, instead needing more extended exposure to acidic stress of 1.5 hours. LipF is annotated to be an esterase and may also function to alter the cell wall structure. LipF has been shown to be part of the two component system *phoP/R* regulon. In fact many genes involved in the PhoP/PhoR regulon including *pks2*, *pks3*, and *pks4* are responsive to acidic stress (Table 1.) (Gonzalo-Asensio et al, 2009; Rohde et al, 2007). Thus PhoP/R may be responding to acidic stress or conversely PhoP/R controls a downstream regulator that responds to acidity. The *ompATb* gene encodes a porin that is active specifically at low pH and functions to pump ammonia into the phagosomal environment which serves to neutralize acidity (Song et al, 2011). Longer term exposure to acidic stress seems to stimulate production of triacylglycerol. *Tgs1* is not upregu‐ lated by short term acid exposure but exposure of three weeks duration or more (Sirakova et al, 2006; Low et al, 2009; Deb et al, 2009). Triacylglycerol production may be important for mycobacteria to resist stress and survive a dormant period which is induced by stress condi‐ tions. An energy source such as triacylglycerol may be needed to reanimate from dormancy once stresses such as acidity are removed. Mutatagenesis studies also revealed genes involved cell wall/cell envelope synthesis when mutated resulted in mycobacteria which were unable to maintain neutral pH within their microbial cytoplasm in the presence of acidic stress (Vandal et al, 2008; Vandal et al, 2009, Biswass et al, 2010).

The type VII secretion system, Esx-1, may also may be involved in response to acid stress (Abdallah et al, 2007). The 6 kDa early secreted antigenic target (Esat-6) and the 10kDa cul‐ ture filtrate protein (CFP-10) are secreted by Esx-1. These two proteins form a heterodimer that can dissociate at acidic pH. Esat-6 is capable of lysing membranes, and *M. tuberculosis* has been identified to reside extraphagosomally in the cytoplasm of macrophages in some cases. In addition when the *esx-1* gene was mutated it could result in an *M. tuberculosis* strain that fails to escape from the phagosomal compartment into the cytoplasm (Simeone et al, 2009). Thus Esat-6 may be involved in mycobacterial responses to acidity and adapta‐ tion to *in vivo* stressors.
