**3.1 Host phagocytes (mouse and human)**

The first work with a whole genome description of *M. tuberculosis* gene expression is that of Schnappinger et al. (Schnappinger et al., 2003). Activated by INF-γ and non-activated mouse macrophages were used in this work as a model system. Rachman et al. characterized the *M. tuberculosis* genes with enhanced expression in activated and inactivated mouse macrophages both relative to each other and to mycobacteria *in vitro* (Rachman et al., 2006). Rohde et al. studied changes in the *M. tuberculosis* transcriptome at the initial stages of infection of mouse macrophages and demonstrated a dynamic enhancement in the expression level of some genes during the first 24 hours post infection (Rohde et al., 2007).

Cappelli et al. were the first to characterize the *M. tuberculosis* whole genome gene expression *in vivo* in human macrophages (Cappelli et al., 2006). Fontan et al. analyzed transcriptomes of *M. tuberculosis* from macrophages of the THP-1 cell line in 4 and 24 hours post infection (Fontan et al., 2008). In the work of Tailleux et al., the authors performed the first comparative analysis of gene expression in *M. tuberculosis* from infected macrophages and dendrite cells (Tailleux et al., 2008). An *in vivo* transcriptome comparison of two differently virulent *M. tuberculosis* strains (H37Rv and H37Ra) was first done by Li et al. (Li et al., 2010).

By the present time, the most extensive study of mycobacterial gene expression *in vivo* is that by Homolka et al. (Homolka et al., 2010). The authors performed a comparative analysis of expression profiles for 3 clinical isolates of *M. africanum*, 12 clinical isolates of *M. tuberculosis*, and two reference laboratory strains (*M. tuberculosis* H37Rv and CDC1551) in activated and non-activated mouse macrophages. This work resulted in the isolation of gene groups whose expression changes irrespective of mycobacterial strain and/or the activation status of the host macrophage.

#### **3.2 Models of** *M. tuberculosis* **infection in laboratory animals**

In 2004, Talaat et al. first performed an analysis of whole genome *M. tuberculosis* gene expression under natural conditions in a living organism (mouse) (Talaat et al., 2004). They studied changes in the pathogen transcriptome composition at different time intervals post infection (7, 14, 21 or 28 days) and for different host genotypes (immunocompetent Balb/c mice and immunodeficient SCID mice). In 2007, Talaat et al. published a paper devoted to the analysis of *M. tuberculosis* gene expression in the lungs of Balb/c mice at later stages of the infection process (Talaat et al., 2007). In 2010, researchers of our group used a new approach to the enrichment of bacterial cDNA for analysis of *M. tuberculosis* gene expression in lung tissues of infected mice (Azhikina et al., 2010). The data obtained by us on quantitative and qualitative composition of the bacterial transcriptome were in good agreement with similar data of the Talaat's group (Talaat et al., 2007).

*Mycobacterium tuberculosis* Transcriptome *In Vivo* Studies –

Rohde et al., 2007).

**4.2 Energy metabolism: Cell respiration** 

et al., 2008; Schnappinger et al., 2003; Shi et al., 2005).

replication (Homolka et al., 2010; Tailleux et al., 2008).

**4.3 Protein biosynthesis and cell growth** 

**4.4 Defense mechanisms, DNA replication** 

Waddell et al., 2004).

A Key to Understand the Pathogen Adaptation Mechanism 133

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;

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

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*

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;

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

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 obtained
