**4.2 Energy metabolism: Cell respiration**

According to data obtained in studies of *in vivo M. tuberculosis* gene expression, energy metabolism of mycobacteria is undergoing a significant transformation during infection process. A characteristic of this transformation is a gradual decrease in the level of the type I NADH dehydrogenase (*nuoA-N*) gene expression and increase in expression of the nitrate reductase gene cluster *narGHJI* and of the *narK2* gene, the product of which is a nitrate transporter protein (Azhikina et al., 2010; Schnappinger et al., 2003; Tailleux et al., 2008). Such a metabolic shift most probably suggests that ETC is being reoriented to the using of nitrate electrons as a finite acceptor. Also, in most cases, aa3 type cytochrome c oxidase (*ctaBECD*) and cytochrome c reductase (*qcrCAB*) gene expression is downregulated (Garton et al., 2008; Schnappinger et al., 2003; Shi et al., 2005).

### **4.3 Protein biosynthesis and cell growth**

Decreased expression of ribosomal protein genes (*rpl*, *rpm*, *rps*) indicates a reduced need for the synthesis of new proteins. Usually this phenomenon occurs in conditions non optimal for the pathogen (dendrite cells, activated macrophages) and correlates with decreased expression level of the ATP-synthase (*atpA-H*) gene and slowdown of *M. tuberculosis* replication (Homolka et al., 2010; Tailleux et al., 2008).

#### **4.4 Defense mechanisms, DNA replication**

The compartment (early phagolysosome) of *M. tuberculosis* residing at persisting in macrophages represents a rather non-aggressive environment with practically no hydrolytic activity and pH 6.2-6.4. Nevertheless, the mycobacteria are under the influence of many stress factors like active forms of oxygen and nitrogen or the apoptotic death of the host cell. The effect of stress factors induces an upregulation of genes of the DNA repair and recombination (*dinF/G*) systems (Rachman et al., 2006; Schnappinger et al., 2003; Talaat et al., 2004), as well as chaperon genes (*groES*, *groEL1/2*, *dnaJ/K*, *hspX*) (Fontan et al., 2008; Garton et al., 2008; Homolka et al., 2010; Karakousis et al., 2004; Rohde et al., 2007; Tailleux et al., 2008). Certain data indicate that this effect is not a specific reaction on intracellular conditions, but part of adaptive response to stress (Boshoff et al., 2004; Waddell et al., 2004).

*Mycobacterium tuberculosis* Transcriptome *In Vivo* Studies –

A Key to Understand the Pathogen Adaptation Mechanism 135

in mouse phagosomes (Rohde et al., 2007), as well as in artificial mouse granulomas and in

Other transcription regulatory genes whose upregulation was observed in *in vivo* experiments are *whiB3* (Fontan et al., 2008; Rohde et al., 2007), *ethR*, *ideR*, *kstR* and *relA*  (Fontan et al., 2008; Schnappinger et al., 2003; Tailleux et al., 2008). In addition, *M. tuberculosis* has 12 two-component regulatory systems (Tucker et al., 2007). Two of them, *phoPR* (Gonzalo-Asensio et al., 2008) and *dosRS* (*devRS*) (Park et al., 2003), were studied more thoroughly than others. It was shown that functional activity of the phoPR system supports the *M. tuberculosis* virulence by regulating the metabolism of complex lipids and the work of the ESX secretion systems (Gonzalo-Asensio et al., 2008). The positive transcription regulator phoP was observed to induce genes under its control in a low-acid (pH 6.4) environment of mouse macrophage phagosomes (Rohde et al., 2007). Not less important is the two component regulatory system dosRS responsible for expression regulation of about 50 genes (Park et al., 2003). Enhanced expression of these genes was repeatedly observed in the course of mycobacterial infection of macrophages (Fontan et al., 2008; Rohde et al., 2007; Schnappinger et al., 2003; Tailleux et al., 2008). Moreover, *dosRS*  regulon genes were expressed practically in all other conditions: in *M. tuberculosis* from artificial mouse granulomas, samples of mouse lung tissues and surgical samples of human lung, as well as in samples of sputum and in some experiments *in vitro* (Garton et al., 2008; Homolka et al., 2010; Karakousis et al., 2004; Li et al., 2010; Talaat et al., 2007; Timm et al., 2003). The functional role of this regulon is still not quite clear, but its activity was suggested to be important for *M. tuberculosis* adaptation to variations in the redox status during the

the mouse lung, respectively (Karakousis et al., 2004; Talaat et al., 2004).

infection process (Bacon et al., 2004; Bacon & Marsh, 2007; Rustad et al., 2009).

**in genetically different mouse models** 

response) to these bacteria (Skvortsov et al., 2010).

aggressive, and the infected mice had a longer survival.

**5. Profiling of** *Mycobacterium tuberculosis* **gene expression during infection** 

We have carried out a comparative study of *M. tuberculosis* transcriptomes in order to reveal the features of expression profiles that correlate with progressing disease, and also to understand the difference between efficient and defective defence mechanisms at the level of bacterial gene expression. To this end, at different stages of the infection process, we performed a comparative quantitative and qualitative analysis of the sequences transcribed during infection of mice sensitive (inefficient immune response) and resistant (efficient

We have compared transcriptomes of *M. tuberculosis* H37Rv in infected mice of two lineages, I/StSnEgYCit (I/St) and C57BL/6JCit (B6). These lineages have been earlier described in detail (Kondratieva et al., 2010), and the B6 lineage was shown to be more resistant to the infection by *M. tuberculosis* than I/St. In particular, the infection process in B6 mice was less

Female mice of both lineages were aerogenically infected with *M. tuberculosis* bacteria. In 4 and 6 weeks post infection, the infected mice were killed, and total lung RNA isolated. Samples of total RNA from lung tissues of I/St and B6 mice were used to synthesize cDNA enriched in fragments of bacterial cDNA using coincidence cloning procedure (Azhikina et al., 2010). As a result, three cDNA libraries were obtained, which represented transcriptomes of *M. tuberculosis* from lung tissues of I/St mice on week 6 post infection
