## 4 Discussion  
Technologies are currently being used to develop new vaccines and/or treatments to prevent TB, which is a global health priority. One of these is the use of proteomics and genetic engineering to discover and produce antigens with a known ability to elicit an immune response [67]. Our work focused on the genetic engineering of mycobacterial strains belonging to the Mtb complex to produce antigens of interest. Such antigens and their epitopes, either alone or in combination, may help to develop new ways to increase the efficacy of current TB vaccines, for example, the currently available _M. bovis_ BCG live-attenuated vaccine [68; 69; 70; 71; 72]. Therefore, we generated and analyzed two recombinant strains of _M. microti_ overexpressing the _M. tuberculosis_ PstS-1 and Ag85B antigens. Both antigens are known to stimulate the immune system and act as potent immunomodulators in the production of cytokines and antibodies when used as multimeric vaccine components [37; 68; 70; 71]. The complete genome of _M. microti_ has not been released up to date. However, studies show that the RD1 region is absent in several _M. microti_ strains [73]. In our work, a comparative genetic analysis of Rv0934 (PstS-1) and Rv1886c (Ag85B) revealed that these genes are present in our wild-type _M. microti_ (ATCC 11152) strain with a homology of 99.9% and 100%, respectively. This confirms that these selected antigens from _M. microti_ are very similar in sequence to those previously described in _M. tuberculosis_ and may have a similar immunomodulatory function. PstS-1 and Ag85B were overexpressed and detected in both _M. microti_ strains (Figure 3). For the PstS-1 antigen, we observed differences in the MW size depending on whether the protein originated from a cell extract or from the secreted form. First, an analysis of the cellular extract of PstS-1 revealed a band showing a 5.2-fold increase in production of the antigen with a MW of \(\sim\)32 kDa (Figure 3, lane 3). However, we did not observe a secretion of PstS-1 in this strain (Figure 3, lane 6). Instead, we detected a band size of 38 kDa in _M. microti_ (wt) and _M. microti_ (ev) (Figure 3, lanes 4 and 5). We hypothesize that this difference in the molecular mass of this protein is due to the detection of proteins prior to the post-translational modifications, as PstS-1 typically undergoes post-translational modifications,  
Figure 5: Venn diagram of the differential expression proteome of the cellular extract from _M. microti_ strains. P.A, P.B and P.C: protein spots from Figure S3A–C, respectively. including glycosylation and lipoylation, which likely contribute to its molecular mass [36]. In addition, the glycosylation of _M. tuberculosis_ proteins may also represent a mechanism for regulating protein export [74]. It is possible that the recombination influenced the transport of this protein outside the bacterial cell. However, this is not a problem, as the intracellular antigen could still be available through the processing performed using professional antigen-presenting cells, where the phagosome maturation leads to the lysis of the microorganism and the antigens are subsequently presented by the molecules of the major histocompatibility complex (MHC) [75; 76; 77]. On the other hand, the expression analysis of Ag85B showed that this antigen was successfully expressed in both the cellular and secreted forms. Western blotting showed the presence of the three bands (Figure 3C lanes 1-6) in both the cell extract and the supernatant. This is probably due to the fact that the antibody used in this study can detect the Ag85 complex (A, B and C) in addition to Ag85B (Figure 3C, lanes 1-6 compared to control lane 7). These results led us to investigate whether the recombinant strains had different proteomes and/or glycosylation profiles. We found that each strain of _M. microti_-PstS-1 and _M. microti_-Ag85B had a different proteomic profile.