## 4 Discussion
We identified six proteins (SodA, hypothetical protein MAP3475c, DapA, MoxR, RpoA and Eno) involved in the energy production process, possibly due to an increased heterologous protein production, that are not expressed in the _M. microti_ wild type. Similar observations were previously made by [PERSON] et al. who reported that the overexpression of proteins in recombinant _Escherichia coli_ strains leads to a change in the protein profiles, resulting in a differential expression of different cellular proteins [78; 79]. There are few studies describing the presence of glycosylation in the proteins of the genus _Mycobacterium_, and most existing studies have focused on mannosylation [54; 80; 81]. Examining the glycosylation profiles in the wild-type and the recombinant _M. microti_ allowed us to identify two potential glycosylation types in these strains: mannosylation and sialylation, as shown in Figure S2. Mannose glycosylation is widely reported in the genus _Mycobacterium_[49; 50; 54; 82]. In the present study, we identified and sequenced eight proteins that were potentially glycosylated with terminal mannose, including MAP_4308c, hypothetical protein p40, MAP_3192, FabG, GlyA, Eno, MAV_0024 and GroEL. The mannosylation has various functions in mycobacteria. For example, it is part of the cell wall and is primarily associated with virulent species and enables protein recognition by mannose and the Toll-like receptor (TLR) 2 expressed by macrophages and other cell types [83]. In addition, studies of proteins that were glycosylated with mannose, such as PstS-1 from _M. tuberculosis_ H37 Rv, have shown that these proteins promote phagocytosis [35]. [PERSON] et al. developed ZXL1 (double-stranded DNA), a therapeutic target that prevents mannose from binding to host cells by inhibiting the immunosuppression of CD11c+ dendritic cells and enhancing the activity of antigen-presenting cells for the activation of naive CD4+ Th1 cells [84]. For PstS-1 and Ag85B, we predicted the glycosylation profiles using three algorithms that allowed us to compare the different results in terms of the number and position of residues involved in mannosylation. Our results show that PstS-1 could be produced in its mannosylated form. In contrast, no glycosylation was predicted for Ag85B (Table 1) using the GLYCOPP V 1.0 algorithm. We have identified proteins that may be glycosylated with sialic acid (Figure S2B). Currently, there is little evidence of proteins glycosylated with sialic acid in the genus Mycobacterium. Therefore, we searched the genome of _M. microti_ for enzymes associated with sialic acid metabolism that have been identified in other bacteria, including _Neisseria meningitides, E. coli_ K12 and _Haemophilus influenzae_[85; 86; 87]. We found that _M. microti_ contains gene encoding proteins that are known to be involved in the de novo synthesis of sialic acid (UDP-\(N\)-acetylglucosamine-pyrophosphary/glucoseamine-1-phosphate-\(N\)-acetyltransferase, L-serine dehydratase and the hypothetical protein B8A25_03135) in the case of _M. tuberculosis sp._ variant _microti_ (data not shown). Overall, these results remain to be confirmed by mass spectrometry assays. The identification of the differences in the proteomic and glycosylation profiles between the wild type and recombinant strains of _M. microti_ may lead to alterations in the immune system recognition and evasion [89; 90]. For example, the mannose glycosylation of the protein APA has been shown to play a key role in its antigenicity, while the deglycosylation of the same protein has been demonstrated to modify the immune response elicited in guinea pigs [53]. Taken together, these results demonstrate the need to thoroughly investigate the proteomic profiles of the different mycobacterial strains being considered for the development of new TB vaccines, as these events may alter the immune response and the effectiveness of these potential vaccines.