**4. Intracellular** *M. tuberculosis* **transcriptome**

The data obtained by different authors in *in vivo* systems allow to single out a number of the main transcriptome features characteristic of mycobacteria persisting in macrophages. Those are primarily changes in expression of the genes involved in pathogen adaptation, as well as of the genes encoding different factors of immune response. In macrophages, mycobacteria are localized within phagosomes which makes a barrier for the immune system components, but, at the same time, complicates the access of the pathogen to nutrients and microelements. Changes in *M. tuberculosis* gene expression are aimed primarily at forming of the environment able to *in vivo* maintain the functional activity of mycobacteria.

### **4.1 Lipid metabolism**

Lipid metabolism is a key process for *M. tuberculosis* which is directly or indirectly confirmed by the presence in the genome of a considerable number of lipid metabolism genes, indispensability of some genes as follows from data of transposon mutagenesis (Sassetti et al., 2003), decrease or lack of virulence observed in *M. tuberculosis* strains mutant for the genes of this functional category (Neyrolles & Guilhot, 2011), and by other factors.

One of the most characteristic changes in the expression of the genes involved in lipid metabolism is activation of the expression of the genes from clusters *fadA*, *fadB*, *fadD*, *fadE* and *echA* (Azhikina et al., 2010; Munoz-Elias & McKinney, 2006; Schnappinger et al., 2003; Tailleux et al., 2008). These genes encode enzymes involved in β-oxidation of fatty acids and the catabolism of cholesterol. The final products of their activity are acetyl-CoA and propionyl-CoA, the participants of the methylcitrate and tricarboxylic acids cycles.

Enhanced expression of the isocitrate lyase gene *icl1* is also related to the methylcitrate cycle. Isocitrate lyase is a key enzyme of the glyoxylate cycle that is activated when the main carbon source are fatty acids. During this process, activated acetate (acetyl-CoA) is being stepwise converted into malate through the stage of glyoxylic acid formation. Malate can be converted into piruvate by the enzymatic activity of the *pckA* gene product, whose enhanced expression was also observed *in vivo* in mycobacteria (Marrero et al., 2010; Schnappinger et al., 2003; Tailleux et al., 2008). Apart from the maintenance of the glyoxylate bypass, the activity of isocitrate lyase is also needed to utilize cytotoxic propionyl-CoA accumulated during the life cycle of mycobacteria (Savvi et al., 2008). This utilization is possible due to the ability of isocitrate lyase to function as 2-methylisocytrate lyase that facilitates the conversion of propionyl-CoA into succinate. Propionyl-CoA can be also metabolized by a conversion into methylmalonyl-CoA and then into succinate, or included into certain components of the cell wall, such as phthiocerol dimycocerosate (PDIM) or sulfolipid-1 (SL-1) (Russell et al., 2010).

The report of Rachman et al. published in 2006 is thus far the only work in which *M. tuberculosis* gene expression was studied directly in the human lungs (surgical samples) (Rachman et al., 2006). In this work, the pathogen's whole genome gene expression profiles from granuloma, pericavitary lung tissue and morphologically normal lung tissue were

The data obtained by different authors in *in vivo* systems allow to single out a number of the main transcriptome features characteristic of mycobacteria persisting in macrophages. Those are primarily changes in expression of the genes involved in pathogen adaptation, as well as of the genes encoding different factors of immune response. In macrophages, mycobacteria are localized within phagosomes which makes a barrier for the immune system components, but, at the same time, complicates the access of the pathogen to nutrients and microelements. Changes in *M. tuberculosis* gene expression are aimed primarily at forming

Lipid metabolism is a key process for *M. tuberculosis* which is directly or indirectly confirmed by the presence in the genome of a considerable number of lipid metabolism genes, indispensability of some genes as follows from data of transposon mutagenesis (Sassetti et al., 2003), decrease or lack of virulence observed in *M. tuberculosis* strains mutant for the genes of this functional category (Neyrolles & Guilhot, 2011), and by other factors. One of the most characteristic changes in the expression of the genes involved in lipid metabolism is activation of the expression of the genes from clusters *fadA*, *fadB*, *fadD*, *fadE* and *echA* (Azhikina et al., 2010; Munoz-Elias & McKinney, 2006; Schnappinger et al., 2003; Tailleux et al., 2008). These genes encode enzymes involved in β-oxidation of fatty acids and the catabolism of cholesterol. The final products of their activity are acetyl-CoA and

of the environment able to *in vivo* maintain the functional activity of mycobacteria.

propionyl-CoA, the participants of the methylcitrate and tricarboxylic acids cycles.

Enhanced expression of the isocitrate lyase gene *icl1* is also related to the methylcitrate cycle. Isocitrate lyase is a key enzyme of the glyoxylate cycle that is activated when the main carbon source are fatty acids. During this process, activated acetate (acetyl-CoA) is being stepwise converted into malate through the stage of glyoxylic acid formation. Malate can be converted into piruvate by the enzymatic activity of the *pckA* gene product, whose enhanced expression was also observed *in vivo* in mycobacteria (Marrero et al., 2010; Schnappinger et al., 2003; Tailleux et al., 2008). Apart from the maintenance of the glyoxylate bypass, the activity of isocitrate lyase is also needed to utilize cytotoxic propionyl-CoA accumulated during the life cycle of mycobacteria (Savvi et al., 2008). This utilization is possible due to the ability of isocitrate lyase to function as 2-methylisocytrate lyase that facilitates the conversion of propionyl-CoA into succinate. Propionyl-CoA can be also metabolized by a conversion into methylmalonyl-CoA and then into succinate, or included into certain components of the cell wall, such as phthiocerol dimycocerosate (PDIM) or sulfolipid-1

**3.3 Examination of the** *M. tuberculosis* **transcriptome in human tissues** 

**4. Intracellular** *M. tuberculosis* **transcriptome** 

obtained

**4.1 Lipid metabolism** 

(SL-1) (Russell et al., 2010).

Among other genes of lipid metabolism expressed predominantly *in vivo*, there are *desA*  genes encoding desaturases of fatty acids (Homolka et al., 2010; Li et al., 2010; Rachman et al., 2006; Schnappinger et al., 2003). The *papA* and *pks* genes, whose protein products are needed for synthesis of polyketides as components of the *M. tuberculosis* cell wall (Bhatt et al., 2007; Hatzios et al., 2009; Sirakova et al., 2001), are practically always transcribed in experiments. However, the level of their transcription *in vivo* varies, possibly reflecting variations in the lipid metabolism depending upon specific conditions (Azhikina et al., 2010; Homolka et al., 2010; Rohde et al., 2007; Tailleux et al., 2008). Interestingly, transcription of these genes is decreased in the avirulent *M. bovis* BCG and *M. tuberculosis* H37Ra strains as compared with the virulent *M. tuberculosis* H37Rv strain (Li et al., 2010; Rohde et al., 2007).
