**5. Profiling of** *Mycobacterium tuberculosis* **gene expression during infection in genetically different mouse models**

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

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 aggressive, and the infected mice had a longer survival.

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

*Mycobacterium tuberculosis* Transcriptome *In Vivo* Studies –

*M. tuberculosis*-specific (unique)

*M. tuberculosis*-specific (unique)

The number of *M. tuberculosis* genes

Genes expressed, % of the total number

sequences

libraries

expressed in each sample.

A Key to Understand the Pathogen Adaptation Mechanism 137

Library CC4(RES) CC6(SUS) CC6(RES) Total sequences read 73410 75655 40966

sequences, % of total sequences read 20,4 57,7 83,6

expressed (at least one reading) 1012 1353 1940

of genes in the library 25,2 33,7 48,3

Table 1. Results of sequencing and mapping of the CC4(RES), CC6(RES) and CC6(SUS)

Of 4012 *M. tuberculosis* genes and 7 pseudogenes, in the CC4(RES) sample 1012 (25.2%), in CC4(SUS) - 1353 (33.7%), and in CC6(RES) – 1940 (48.3%) genes were expressed. 1428 (35,5%) genes were not expressed in either of the samples, whereas 469 (11.7%) genes were

The distribution of the expressed genes between functional categories is shown in Fig. 3.

Fig. 3. Assignment to biochemical categories of the *M. tuberculosis* H37Rv genes expressed in samples of lung tissue of B6 mice on weeks 4 and 6 post infection and of I/St mice on week 6

The comparison of *M. tuberculosis* transcriptomes during infection in a genetically stable mouse lineage (СС6(RES) vs CC4(RES)) and in genetically different mice (СС6(RES) vs CC6(SUS)), described above, was aimed at the search of genes whose expression is enhanced in the course of infection, specifically in B6 mice on week 6 as compared with

post infection (CC4(RES), CC6(RES) and CC6(SUS) libraries, respectively)

**5.1 Genes whose expression is enhanced during infection** 

Mobile elements (insertion sequences and phages) were excluded from the analysis.

14990 43618 34234

(СС6(SUS)) and from lung tissues of B6 mice on weeks 4 and 6 post infection (СС4(RES) and СС6(RES), respectively). The libraries were analyzed using the 454 pyrosequencing technology. A general scheme of the experiment is shown in Fig. 2, and general characteristics of the libraries analyzed are presented in Table 1. In total, sequences of 190031 cDNA fragments were determined: 73410 from CC4(RES), 75655 from CC4(SUS), and 40966 from CC6(RES).

Fig. 2. Scheme of the transcriptomes comparison experiment. Res – genetically resistant mice, Sus – genetically susceptible mice, CC – library enriched with bacterial cDNA

Using a standalone BLASTn algorithm, the nucleotide sequences obtained were mapped to the genome sequence of *M. tuberculosis* H37Rv (GenBank version: AL123456.2). The mapping revealed that the CC4(RES), CC6(SUS) and CC6(RES) samples contained 14990 (20.42%), 43618 (57.65%) and 34234 (83.57%) *M. tuberculosis* sequences, respectively.

The results obtained demonstrate that the technology used allowed us to considerably enrich the cDNA samples with bacterial sequences.

(СС6(SUS)) and from lung tissues of B6 mice on weeks 4 and 6 post infection (СС4(RES) and СС6(RES), respectively). The libraries were analyzed using the 454 pyrosequencing technology. A general scheme of the experiment is shown in Fig. 2, and general characteristics of the libraries analyzed are presented in Table 1. In total, sequences of 190031 cDNA fragments were determined: 73410 from CC4(RES), 75655 from CC4(SUS), and

Fig. 2. Scheme of the transcriptomes comparison experiment. Res – genetically resistant mice, Sus – genetically susceptible mice, CC – library enriched with bacterial cDNA

(20.42%), 43618 (57.65%) and 34234 (83.57%) *M. tuberculosis* sequences, respectively.

enrich the cDNA samples with bacterial sequences.

Using a standalone BLASTn algorithm, the nucleotide sequences obtained were mapped to the genome sequence of *M. tuberculosis* H37Rv (GenBank version: AL123456.2). The mapping revealed that the CC4(RES), CC6(SUS) and CC6(RES) samples contained 14990

The results obtained demonstrate that the technology used allowed us to considerably

40966 from CC6(RES).


Table 1. Results of sequencing and mapping of the CC4(RES), CC6(RES) and CC6(SUS) libraries

Of 4012 *M. tuberculosis* genes and 7 pseudogenes, in the CC4(RES) sample 1012 (25.2%), in CC4(SUS) - 1353 (33.7%), and in CC6(RES) – 1940 (48.3%) genes were expressed. 1428 (35,5%) genes were not expressed in either of the samples, whereas 469 (11.7%) genes were expressed in each sample.

The distribution of the expressed genes between functional categories is shown in Fig. 3. Mobile elements (insertion sequences and phages) were excluded from the analysis.

Fig. 3. Assignment to biochemical categories of the *M. tuberculosis* H37Rv genes expressed in samples of lung tissue of B6 mice on weeks 4 and 6 post infection and of I/St mice on week 6 post infection (CC4(RES), CC6(RES) and CC6(SUS) libraries, respectively)

#### **5.1 Genes whose expression is enhanced during infection**

The comparison of *M. tuberculosis* transcriptomes during infection in a genetically stable mouse lineage (СС6(RES) vs CC4(RES)) and in genetically different mice (СС6(RES) vs CC6(SUS)), described above, was aimed at the search of genes whose expression is enhanced in the course of infection, specifically in B6 mice on week 6 as compared with

*Mycobacterium tuberculosis* Transcriptome *In Vivo* Studies –

**mechanisms** 

http://tuberculist.epfl.ch)

even *M. tuberculosis*.

antigenic variability

state of latent infection.

suggest a forced usage of lipids as the source of energy and carbon.

A Key to Understand the Pathogen Adaptation Mechanism 139

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

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

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,

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

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

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

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 search on *M. tuberculosis* genes:


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)
