**7. Response to oxidative damage**

The ability of *M. tuberculosis* to survive in a dormant state relies on maintaining cell integrity, viability, and a proton motive (Rustad et al, 2008). Entry into a dormant state may be followed later by reactivation and growth of this microorganism, and may occur due to waning immunity, age, or disease. T-cells originally controlling infection may become less activated and numbers of T-cells may decrease allowing mycobacteria increased ease of replication in host macrophages. *M. tuberculosis* needs energy to exit this dormant phase, and this may be found in the form of triacylglycerol which is known to accumulate in response to acidic stress, nitric oxide exposure, and lowered oxygen tension (Table 1.) (Sirakova et al, 2006; Garton et al, 2008). In fact triacylglycerol has been shown to be important to transition from dormancy to active growth (Low et al, 2009). The highly pathogenic strain of *M. tuberculosis*, the Beijing lineage strain, over produces triacylglycerol perhaps giving the microorganism a competitive

*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

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

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

edge in resisting hypoxic stress and dormancy (Fallow et al, 2010).

6 Tuberculosis - Current Issues in Diagnosis and Management

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

environmental cousins (Kirschner et al, 1992; Kirschner et al, 1999).

of the infection.

Inside phagosomes of activated macrophages tubercle bacilli are exposed to reactive oxygen intermediates. *M. tuberculosis* traffics to phagosomes, and a large number of genes are upre‐ gulated by oxidative stress indicating this is an important stress *in vivo* (Wu et al, 2007). In addition nutrients are limited in the phagosome which may cause *M. tuberculosis* to enter a stationary phase of growth, which has been shown to induce internal oxidative damage. The gene *whiB1* is more active during stationary phase, and the protein produced by this gene has been shown to reduce cellular disulphide bridges that may predominate during this adapta‐ tional phase (Garge et al, 2009).

iron to be bound by the host cellular factor ferritin. Infection and inflammation are natural signals to the host to limit availability of iron. Proinflammatory cytokines stimulate hepcidin production, decrease iron uptake from the gut, and inhibits the iron efflux protein ferroportin (Johnson and Wessingling-Resnick, 2012). Inflammation thus inhibits iron uptake by the intestinal epithelium thus preventing iron from being loaded onto transferrin. Interfering with uptake limits iron availability in the host, and *M. tuberculosis* has been shown to be severely growth restricted in a low iron environment. It has been demonstrated in African studies that iron supplementation increases incidence of tuberculosis. Thus being anemic may be protec‐ tive against infectious processes. Within human macrophages, Nramp1 (natural resistance associated macrophage protein) is produced and localizes to the phagosomal compartment where it reduces iron within this site possibly by extrusion. This function confers resistance to *M. tuberculosis* infections and mutations in the *nramp1* gene can result in increased suscepti‐ bility to active disease due to *M. tuberculosis* infection (Johnson and Wessingling-Resnick,

*Mycobacterium tuberculosis* Adaptation to Survival in a Human Host

http://dx.doi.org/10.5772/54956

9

Mycobacteria have a variety of systems which aid in the uptake of iron and the regulation of iron responsive genes. As mycobacteria have been shown to be somewhat novel among gram positive bacteria, they possess an outer mycolic acid based membrane, as well as an inner membrane and periplasmic space. Porins in the outer membrane appear to transport iron in the presence of high iron conditions (Jones and Niederweis, 2010). *M. tuberculosis* under low iron conditions can produce the siderophore carboxymycobactin as well as my‐ cobactin (Table 1.) (Banerjee et al, 2011). These molecules bind with a higher affinity to iron than the human host's storage proteins and steal iron from the host. Mycobactin is present within the inner membrane and thus can only bind iron imported into the periplasmic space. Interestingly lipid membranes with associated mycobactins may diffuse out, travel to lipid vesicles in the host cell, and sequester iron. These structures may recycle back to in‐ teract with the mycobacterium. Disruption of the genes responsible for production of my‐ cobactins can cause these mutant mycobacteria to replicate less well in macrophages (Banerjee et al, 2011). Carboxymycobactins are excreted possibly by the type VII secretion or ESX system. Externally the carboxymycobactins bind available iron from transferrin (Banerjee et al, 2011). Porins and also ABC transporters may allow import of these iron loaded carboxymycobactins (Banerjee et al, 2011). The host cell, in response to infection and inflammation, produces siderocalins such as lipocalin-2 that can bind to and inactivate my‐ cobactin from *M. tuberculosis* thus interfering with mycobacterial iron acquisition (Johnson and Wessingling-Resnick, 2012). In fact mice deleted for genes involved in production of siderocalin are much more susceptible to mortality due to *M. tuberculosis* infection (Johnson and Wessingling-Resnick, 2012). Inside the mycobacterial cell, iron is stored in bacterioferri‐ tin and a ferritin like protein. These proteins are required for replication in human macro‐ phages and guinea pigs, act to store iron, and also to limit excess iron in the cells that can lead to iron mediated oxidative damage due to the Fenton reaction (Reddy et al, 2011).

Iron responsive genes in *M. tuberculosis* are controlled in part by the iron dependent regulator IdeR. This protein can act both as an activator and a repressor depending on where it binds within a mycobacterial promoter region (Manabe et al, 1999; Banerjee et al, 2011). Within

2012).

Mycobacteria contain a unique substance, mycothiol, which combats oxidative stress. Other bacterial species utilize glutathione which can also neutralize oxidative stress. Mycothiol contains cysteine residues which are oxidized when that condition predominates thus forming disulfide bonds, creating mycothione, and preventing other molecules in the mycobacterial cell from becoming oxidized (Table 1.). Human cells produce glutathione to combat oxidative damage, and glutathione is toxic to mycobacterial cells perhaps due to a redox imbalance generated by this substance in the mycobacteria (Venketaraman et al, 2008; Connell et al, 2008)). Mycobacteria also contain other molecules to detoxify oxidative damage including superoxide dismutase (SOD) and catalase (KatG) which can inactivate superoxide (Table 1.) (Shi et al, 2008). SOD and KatG are upregulated early in infection indicating an increase in oxidative damage due to superoxide. Oxidative damage is capable of harming DNA, and histone like proteins (LSR2) can protect against damage by compacting DNA and acting as a physical barrier. UvrB which repairs mycobacterial DNA damage also protects against oxidative damage (Darwin and Nathan, 2005; Colangeli et al, 2009).
