**5.1.1 Genes upregulated only in one of genetically different hosts**

We found 17 genes with enhanced expression in CC6(RES) vs CC4(RES) and 44 genes in CC6(RES) vs CC6(SUS). Such a statistics probably reflects the fact that in the first case bacterial genes are expressed in one and the same microenvironment, whereas in the second case microenvironments are different resulting in a greater number of genes upregulated.

Genes, the expression of which is enhanced in the CC6(RES) sample only relative to CC4(RES), mostly belong to categories of cell wall and cell processes, intermediary metabolism and respiration, and lipid metabolism. The protein products of 12 out of 17 genes were detected in a fraction of cell membrane or cell wall where they mainly fulfil transport and defence functions. For example, the *embA* gene codes for indolylacetylinositol arabinosyltransferase EmbA involved in the synthesis of arabinan, and mutations in this gene cause resistance to ethambutol. Also, the *Rv3273* gene encodes carbonate dehydratase that participates in sulfate transport (TubercuList, http://tuberculist.epfl.ch). In the analysis performed, we failed to detect metabolic pathways specifically activated at later infection times as compared to early stages.

Comparing the bacterial transcriptomes within different hosts we found greater variety of biochemical pathways. An increase in energy exchange is reflected in enhanced expression of the genes of three NADH-dehydrogenase subunits (*nuoH*, *nuoI*, *nuoL*), as well as in increased activity of the tricarbonic acid cycle and in upregulation of the *Rv1916* gene. The *Rv1916* gene is the second part of the *aceA* (*icl2*) gene which in *M. tuberculosis* H37Rv is divided into two modules, *Rv1915* and *Rv1916* (*aceAa* и *aceAb*), each of which can be expressed independently. Among other important differences, one can highlight enhanced expression of genes, the products of which are responsible for lipid and amino acid metabolism and catabolism (*lipV*, *lipF*, *Rv2531c*), and genes of DNA repair enzymes (*recO*, *recB*).

other time points. The comparison of СС6(RES) vs CC4(RES) allowed to reveal 226 genes upregulated in infected tissues of B6 mice. A similar comparison of СС6(RES) vs CC6(SUS) revealed 253 genes upregulated in the CC6(RES) sample (Fig. 4). We concentrated our

2. with expression upregulated irrespective of genetic features of the host organism. These genes represent a kind of basic set of genes responsible for a universal compensatory reaction of *M. tuberculosis* to unfavourable conditions of the environment. They are

**17 209 44**

**CC6(RES) vs CC4(RES) CC6(RES) vs CC6(SUS)**

We found 17 genes with enhanced expression in CC6(RES) vs CC4(RES) and 44 genes in CC6(RES) vs CC6(SUS). Such a statistics probably reflects the fact that in the first case bacterial genes are expressed in one and the same microenvironment, whereas in the second case microenvironments are different resulting in a greater number of genes upregulated. Genes, the expression of which is enhanced in the CC6(RES) sample only relative to CC4(RES), mostly belong to categories of cell wall and cell processes, intermediary metabolism and respiration, and lipid metabolism. The protein products of 12 out of 17 genes were detected in a fraction of cell membrane or cell wall where they mainly fulfil transport and defence functions. For example, the *embA* gene codes for indolylacetylinositol arabinosyltransferase EmbA involved in the synthesis of arabinan, and mutations in this gene cause resistance to ethambutol. Also, the *Rv3273* gene encodes carbonate dehydratase that participates in sulfate transport (TubercuList, http://tuberculist.epfl.ch). In the analysis performed, we failed to detect metabolic pathways specifically activated at later infection

Comparing the bacterial transcriptomes within different hosts we found greater variety of biochemical pathways. An increase in energy exchange is reflected in enhanced expression of the genes of three NADH-dehydrogenase subunits (*nuoH*, *nuoI*, *nuoL*), as well as in increased activity of the tricarbonic acid cycle and in upregulation of the *Rv1916* gene. The *Rv1916* gene is the second part of the *aceA* (*icl2*) gene which in *M. tuberculosis* H37Rv is divided into two modules, *Rv1915* and *Rv1916* (*aceAa* и *aceAb*), each of which can be expressed independently. Among other important differences, one can highlight enhanced expression of genes, the products of which are responsible for lipid and amino acid metabolism and catabolism (*lipV*, *lipF*, *Rv2531c*), and genes of DNA repair enzymes

Fig. 4. Venn diagram illustrating the number of upregulated mycobacterial genes in comparisons CC6(RES) vs CC4(RES) (dotted) and CC6(RES) vs CC6(SUS) (solid)

**5.1.1 Genes upregulated only in one of genetically different hosts** 

1. with expression upregulated only in one of genetically different hosts;

termed CUG –Commonly Upregulated Genes.

search on *M. tuberculosis* genes:

times as compared to early stages.

(*recO*, *recB*).

Such a picture is quite predictable as the microenvironment in a resistant host is a hostile habitat which can explain the need in more active repair systems. Increased gene expression of lipolytic enzymes (*lipF*, *lipV*, *plcA*), enzymes of the tricarbonic acid cycle and *aceAb* may suggest a forced usage of lipids as the source of energy and carbon.

#### **5.1.2 CUGs – genes needed for** *M. tuberculosis* **adaptation to different host defense mechanisms**

We have revealed 209 genes upregulated in both comparisons. According to the results of transposon mutagenesis, the products of 44 out of these genes are essential in *M. tuberculosis* (Sassetti et al., 2003), the list of these genes is given in Table 2. *Rv3569c*, *Rv3537*, and *Rv3563*  were earlier shown to be essential for survival in mouse macrophages (TubercuList, http://tuberculist.epfl.ch)

A bit less than one third of the 209 genes belong to the conserved hypothetical (59 genes) and unknown (2 genes) categories. In spite of unknown functions, the genes of these categories may be considered potential therapeutic targets, since their low homology to genes of other microorganisms suggests that they are characteristic just of mycobacteria or even *M. tuberculosis*.

The function of the PE/PPE family proteins is not quite clear, but they are suggested to be needed for antigenic variability in mycobacteria (Karboul et al., 2008). Nevertheless, the *Rv0152c* and *Rv0355c* genes had a high expression level in the CC6(RES) sample, and they were also expressed in the CC4(RES) and CC4(SUS) samples. *Rv3135* encodes a protein essential in *M. tuberculosis* H37Rv that may suggest some additional functions beyond antigenic variability

One more feature of CUGs is an increased activity of amino acid metabolism pathways. It is not clear if the stimulation of this metabolism enzyme expression is due to the absence of available amino acids (and the necessity of their synthesis) or their availability (and the possibility to use them). Poor nutrient conditions of the environment are supported by a high level gene expression of various systems of acquisition and accumulation of nutrients, e.g. such as phosphate (*pstS1*) or iron (*irtA*, *mbtC*, *mbtE*, *mbtF*). A shortage of phosphate is indicated by enhanced expression of the *senX3* gene, a sensor component of the senX3 regX3 two-component system that activates the so called stringent response under phosphorus deficiency. The expression of lipid metabolism genes (fadD, fadE, lipU, *lipJ*) suggests a switch to using lipids as a major source of energy and carbon. Enhanced expression of the *narH* and *narK3* genes implies a switch to anaerobic respiration characteristic of latent infection. Finally, the *secA2* gene is also worth mentioning. This gene codes for translocase SecA2 which is a component of the *M. tuberculosis* secondary transport system Sec that provides for, in particular, secretion of superoxide dismutase SodA and catalase katG. A live vaccine based on an *M. tuberculosis* mutant for the *secA2* gene (Hinchey et al.,2011) showed high efficiency and safety in animal trials. Summarizing, it can be said that CUGs reflect characteristic features of infection in a mouse model. An exception is increased expression of the *atpF* and *atpH* genes, although, according to some reports, their expression decreases in the course of infection as energy demand of the pathogen goes down upon transition to the state of latent infection.

*Mycobacterium tuberculosis* Transcriptome *In Vivo* Studies –

Gene Name Function Description

*Rv3627c Rv3627c* CH hypothetical protein

A Key to Understand the Pathogen Adaptation Mechanism 141

*Rv3515c fadD19* LM Probable fatty-acid-coA ligase fadD19 (fatty-acid-coa

arabinosyltransferase

synthetase) (fatty-acid-coa synthase)

*Rv3411c guaB2* IMaR inositol-5-monophosphate dehydrogenase

*Rv3658c Rv3658c* CWaCP Probable conserved transmembrane protein *Rv3793 embC* CWaCP Integral membrane indolylacetylinositol

processes; LM – lipid metabolism; IP – information pathway; VDA – virulence, detoxification,

adaptation; PE/PPE – PE/PPE protein families; CH – conserved hypotheticals

Table 2. CUGs, found as essential according (Sassetti et al., 2003)

**6. Conclusion** 

colonization of the host.

**7. Acknowledgment** 

**8. References** 

diagnostics and treatment of tuberculosis.

Sciences, and by RBRF grant # 11-04-01325.

*Rv3799c accD4* LM Probable propionyl-CoA carboxylase beta chain 4 RP – regulatory proteins; IMAP – intermediate metabolism and respiration; CWaCP – cell wall and cell

Infectious diseases caused by intracellular pathogenic bacteria represent a significant challenge to health care. The course of the infection depends not only on the protective mechanisms, but also on the specific expression of bacterial genes. Altered expression as a response to the immune reaction of the host organism is critical for the survival and functioning of pathogenic bacteria. Understanding of *M. tuberculosis* transcriptional responses to different stimuli and aggressiveness of the environment gives the opportunity to describe the adaptation mechanisms necessary for bacterial successful survival and

*M. tuberculosis* transcription profiling obtained in different conditions allows to define the core set of adaptive genes (we called it "commonly upregulated genes"), which characterize different phases of *M. tuberculosis* intracellular life – from primary infection through latency to reactivation. The expression of these genes can be considered as a universal reaction of mycobacteria to various stress factors of the environment. Accumulation and analysis of such data is the surest way to proceeding and developing effective approaches towards

The authors thank Dr. Boris Glotov for critically reading the manuscript. The work was supported by grants of the "Leading Scientific Schools of Russia" (project NSh 5638.2010.4), "Molecular and Cellular Biology" program of the Presidium of the Russian Academy of

Abdallah, A. M., Gey van Pittius, N. C., Champion, P. A., Cox, J., Luirink, J.,

Vandenbroucke-Grauls, C. M., Appelmelk, B. J., & Bitter, W. (2007). Type VII

*Rv3593 lpqF* CWaCP Probable conserved lipoprotein lpqF



RP – regulatory proteins; IMAP – intermediate metabolism and respiration; CWaCP – cell wall and cell processes; LM – lipid metabolism; IP – information pathway; VDA – virulence, detoxification, adaptation; PE/PPE – PE/PPE protein families; CH – conserved hypotheticals

Table 2. CUGs, found as essential according (Sassetti et al., 2003)
