## 3 Results
### Differential Proteome of M. microti Recombinant Strains  
Based on the differences in the PstS-1 production and secretion (Figure 3A, lanes 3 and 6), we decided to perform a proteomic analysis to investigate what other changes might occur in native proteins in the _M. microti_-PstS-1 and _M. microti_-Ag85B recombinant strains and compare them with the _M. microti_ wild type. The two-dimensional fluorescence differential gel electrophoresis was performed with labelled proteins from the cell extracts of the three strains, as mentioned in materials and methods. On the fluorescence images obtained for each strain, we observed different protein spots that were differentially expressed compared to each other: _M. microti_ vs. _M. microti_-PstS-1, _M. microti_ vs. _M. microti_-Ag85B and _M. microti_-PstS-1 vs. _M. microti_-Ag85B (Figure S3). The image analysis allowed us to perform a silver stained 2D SDS-PAGE (Figure 4) to identify the spots that were differentially expressed and to select the spots with the highest intensities for further sequencing using MS/MS. Our analysis results showed that 46 proteins were differentially expressed in the three examined _M. microti_ strains and based on intensity, we selected 18 proteins for further analysis. These results suggest that changes in the genetic load of _M. microti_, such as the insertion of foreign plasmids and the subsequent overexpression of proteins, cause changes in the proteome profile of _M. microti_-PstS-1 and _M. microti_-Ag85B in the cell extract. However, further high throughput proteome and transcriptome analyses are still required to substantiate our results.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Antigen** & **UniProt** & **NetOGlyc-4.0 1** & **Glycosylation Predictor** & **GLYCOPP V 1.0 3** & **Experimental** \\  & **Acc. No.** & **NetOGlyc-4.0 1** & **GPP 2** & **GLYCOPP V 1.0 3** & **Determination** \\ \hline \multirow{4}{*}{PstS-1 (Rv0934)} & \multirow{4}{*}{B2 MVV3} & \multirow{4}{*}{B2 MVV3} & T7, T13, S26, S30, S32, T44, & \multirow{4}{*}{T86, S88, T**160**, **T331**} & \multirow{4}{*}{ND} \\  & & **S30**, S32, T35, T41, & & & \\  & & **T44**, T45, S48, S49, & & & \\  & & **S290** & **T160**, S186, T196, T216, T236, & \multirow{4}{*}{**T331**} & \multirow{4}{*}{ND} \\  & & & **S290**, S299, **T331**, S365, T371, & & \\  & & & S373, S374 & & \\ \hline Mod. AA & & 9 & 29 & 4 & \\ \hline \multirow{4}{*}{\
\begin{tabular}{c} Ag85B \\ (Rv1866c) \\ \end{tabular} } & \multirow{4}{*}{P9 WQP1} & \multirow{4}{*}{None predicted} & T2, S5, S42, S57, N71, S116, & \multirow{4}{*}{None predicted} & \multirow{4}{*}{ND} \\  & & S117, S120, S150, T158, S160, & & \\  & & S166, S171, S202, T234, S320, & & & \\  & & & S321 & & \\ \hline Mod. AA & & 0 & 17 & 0 & \\ \hline \multirow{4}{*}{
\begin{tabular}{c} APA \\ (Rv1860) \\ \end{tabular} } & \multirow{4}{*}{P9 WIR7} & \multirow{4}{*}{P9 WIR7} & **T10, T11, S14, S17, T18,** & \multirow{4}{*}{T10, T18, T27, T39, T108, **T1827**, **T27**, S101, S105, T107, **T108**, **T107, **T108**, **T142, S144, S152, T164, S181, & \multirow{4}{*}{T10, T18, T27, T277} \\  & & S243, T274, T276, T277, & & & \\  & & T279, T283 & & & \\  & & & **T274, T276, T283** & & \\ \hline Mod. AA & & 14 & 23 & 6 & 4 \\ \hline \hline \end{tabular}  
* Acc. No.: Accession number, Mod. AA: Modified amino acids, ND: Not determined. APA antigen was used for comparison purposes and analysed in silico without the signal peptide composed of residues 1–39. Residues in bold are shared by two or more prediction tools. 1 Based on a neural network predictions of mucin type GaNAc O-glycosylation sites in mammalian proteins where a value higher than 0.5 was considered a positive hit [61]. 2 Prediction based on random forests algorithms [63]. 3 Prediction based on Composition Profile of Patterns (CPP) where a value higher than 0.9 was considered a positive hit [62].  
\end{table}
Table 1: Potential glycosylated residues in recombinant antigens PstS-1 and Ag85B.