## 3 Results
### Glycoslation of Proteins Produced by M. microti Recombinant Strains  
To determine whether _M. microti_-PstS-1, _M. microti_-Ag85B and the _M. microti_ wild type can still produce glycosylated proteins, we performed a lectin blot analysis using a DIG Glycan Differentiation Kit(tm) (Merck(tm)). As suggested by our results, the recombinant _M. microti_ strains produced glycosylated proteins (Figure S2A). These proteins are likely mannosyllated, as we observed signal recognition with GNA lectin. On the other hand, to our surprise, we detected faint bands with an approximate MW of 50 kDa in the three strains when the lectin blot was performed with MAA lectin, which recognizes sialic acids (Figure S2B). To our knowledge, this is the first time that sialic acid has been detected in proteins produced by _M. microti_ strains. However, a more in-depth analysis of the glycoprotein of _M. microti_, such as MS/MS and the complementation with other techniques, is required to confirm our results. To better understand the glycosylation profile of the PstS-1 and Ag85B antigens, we performed an in silico analysis to predict the potential glycosylation sites on both the recombinant antigens, using three different algorithms to compare the results. In Table 1, we summarize our results of the in silico analysis for the _O_-glycosylation of the PstS-1 and Ag85B proteins. Figure 2: Kinetic growth of _M. microti_ strains in Sautton media. The biomass accumulation in absorbance units (A.U.) measured by optical density (O.D.) during 196 h: (**A**) Lincal scale growth and (**B**) Log scale growth of the strains _M. microti_ (closed circles), _M. microti_-PstS-1 (closed triangles) and _M. microti_-Ag85B (open squares). The red asterisk indicates the collection time for protein analysis. As our results show, we were able to predict the glycosylation sites of PstS-1 and Ag85B and compare them with the alanine and proline rich antigen APA. PstS-1 showed a positive glycosylation profile after all three analyses. However, differences were observed with respect to the glycosylated residues, as can be seen in Table 1. On the other hand, Ag85B showed positive glycosylation sites only with the Glycosylation Predictor GPP algorithm, with 17 hits. When analyzing the antigen APA and its mannosylation profile with the three algorithms, we also found differences in the number and location of the residue. The mannosylation profile of APA was determined by [PERSON] et. al. (1996) [65] and later by [PERSON] et. al. (2013) [29], who together reported seven mannosylated residues (Thr 10, 18, 27, 274, 276, 277 and 279). In our results, we found that the most accurate analysis was performed by the GLYCOPP V 1.0 algorithm with six potentially glycosylated residues (Ser 17, Thr 18, 27, 274, 276 and 277). So far, no glycosylation has been demonstrated for Ag85B by other authors, and this was confirmed by our results with GLYCOPP V 1.0. Although PstS-1 has previously been described as a glycolipoprotein, its mannosylation profile has not previously been determined. Our results with GLYCOPP V 1.0 indicate that Thr 86, 160, 331 and Ser 88 are likely to be mannosylated in vivo. Based on our analysis using the antigen APA as a reference for the glycosylation profile, we suggest that prediction of glycosylation with the GLYCOPP V1.0 algorithm could be used to assess a glycosylation profile similar to that occurring in the glycoproteins produced by mycobacteria. Figure 3: Protein analysis of PstS-1 and Ag85B, either cellular or secreted, produced by _M. microti_ strains. (**A,C**) detection by Western blot of antigens PstS-1 and Ag85B, respectively. (**B,D**) fold expression analysis of PstS-1 and Ag85B, respectively. (**A,C**) lanes 1 and 4: _M. microti_ wild type (wt), lanes 2 and 5: _M. microti_ with empty vector (ev), lane 7 purified protein control. (**A**) lanes 3 and 5 _M. microti_-PstS-1; (**B**) lanes 3 and 5 _M. microti_-Ag85B. The fold change analysis performed by densitometric analysis by Image J. Red asterisk indicates the absence of protein.