**5.** *Mycobacterium tuberculosis* **and dormancy**

efficient at killing or retarding growth of invading microbes. Initially bacilli are taken up into phagosomal compartments and may replicate. As the immune system becomes activated, macrophages are stimulated with INF-γ to increase their efficiency of mycobacterial killing, becoming more efficient at producing reactive oxygen intermediates and acidic stress. In response, *M. tuberculosis* pushes back against the macrophages and differentially regulates key genes. Within macrophages *M. tuberculosis* increases its lipid metabolism which may reflect an environment in the phagosome which lacks available carbohydrates (Table. 1). In addition the enzyme isocitrate lyase (*icl*) is strongly induced *in vivo,* and *icl* is upregulated in all macrophage models. Icl is a key enzyme in the glyoxylate shunt and utilizes fatty acids as an energy source. When *icl* and other genes in the glyoxylate shunt are mutated this results in attenuation *in vivo*. In addition within macrophages, genes involved in stress responses, cell wall component production, anaerobic respiration, siderophore production to scavange iron, diverse sigma factor production, and tranposases that may mutate the genome are all upre‐

Once infection has progressed, tubercle bacilli replicate within incompletely activated mac‐ rophages. Additional macrophages arrive to the site of infection, and engulf newly liberat‐ ed mycobacteria. The immune cells, T-cells, arrive to this location and an immune structure, the granuloma, composed of macrophages and a mantel of T-cells develops. If the host is resistant, and can robustly activate the body's macrophages, then *M. tuberculosis* infection is likely controlled. If the host immune system is weak, or is weakened, *M. tuber‐ culosis* can replicate in the incompletely activated macrophages. Genes of *M. tuberculosis* re‐ quired to resist macrophages will be important in resisting the environment of the granuloma as well. As the infection progresses in susceptible individuals, the centers of the granulomas degenerate and form a caseous, or cheesy, center. At the heart of this is an ele‐ vated lipid metabolism of the host that produces a variety of lipids including cholesterol, cholesteryl esters, triacyglycerol and others (Kim et al, 2010). Interestingly *M. tuberculosis* infection has been shown to induce elevated lipid metabolism in the host (Table 1.). The cell wall lipid of *M. tuberculosis*, trehalose dimycolate or cord factor, induces a granuloma‐ tous response in mice, and this was accompanied by foam cell formation which contains elevated lipids (Kim et al, 2010). It is intriguing to speculate that *M. tuberculosis* infection can induce elevated host lipid metabolism, and as discussed previously as part of adapta‐ tion to *in vivo* growth, *M. tuberculosis* also switches to lipid metabolism and lipids as a pre‐ ferred carbon source (Eisenreich et al, 2010). Thus *M. tuberculosis* induces the host to

gulated (Schnappinger et al, 2003, Beste et al, 2007, Ward et al, 2010).

produce what the microbe has evolved to utilize as an energy source.

Later in infection caseating granulomas continue to breakdown. At a certain point these granulomas begin to liquefy, and host lipases and proteases are present which damage host

**4. Liquefied lesions and sputum**

**3. Adaptation to granulomas and caseation**

4 Tuberculosis - Current Issues in Diagnosis and Management

One third of the world's population is infected with *M. tuberculosis* in part because it causes a latent or dormant infection in a majority of those infected. If therapies are to be developed which can eradicate *M. tuberculosis,* a better understanding of dormancy is required. *M. tuberculosis* can persist for decades in a dormant state within hypoxic granulomas in the lung. Studies have suggested that in a dormant state *M. tuberculosis* is occupied mainly with maintaining cell wall integrity, membrane potential, and protecting its DNA structure. The mycobacterium must also resist the host's immune system. A number of *in vivo* and *in vitro* models have been used to investigate dormancy. These models include exposing mycobacteria to environments that are likely encountered within the host. In one model cultures are stirred slowly and sealed so that oxygen is gradually consumed. In another model nutrient starvation of the bacteria may induce dormancy. In addition, infection of mice, partial treatment with antibiotics, and exposure to immune suppression can lead to dormancy and reactivation (Murphy and Brown, 2007).

The gene encoding a transcriptional regulator, *dosR* (*devR*), part of a two component system that responds to low oxygen seems to be very important in a shift from replicating *M. tuberculosis* to a nonreplicating form (Table 1.). Carbohydrate limitation also upregulated *dosR* and there is indeed an overlap of genes upregulated in phagosomes of macrophages and low carbohydrate availability. In dormancy models aerobic respiratory metabolism was down regulated while anaerobic respiration was upregulated as were DosR controlled genes (Murphy and Brown, 2007). Amino acid and carbon starvation results in the activation of the stringent response. RelA (Rv2583c) mediates this stringent response in *M. tuberculosis* and can globally down regulate components necessary in protein translation, and thus conserve badly needed resources in the mycobacterium during times of stress. RelA may be a target to prevent *M. tuberculosis* from entering dormancy or a target to force *M. tuberculosis* out of dormancy (Murphy and Brown, 2007).

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

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*.

*Mycobacterium tuberculosis* Adaptation to Survival in a Human Host

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

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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

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‐

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‐

et al, 2008; Vandal et al, 2009, Biswass et al, 2010).

tion to *in vivo* stressors.

**7. Response to oxidative damage**
