Resumo

Previous studies regarding C. pseudotuberculosis (Cp) structural proteins and antigenic analysis were mainly performed through in vitro experiments using bacterial defined media. However, when infecting a host, bacterial growth conditions can be different, which can lead to differences at virulence factors expression. In this study, we investigated the expression of Cp cell wall glycoconjugates when grown in animal serum, in comparison to a commercial medium. A high virulent Cp strain (VD57) was grown in Bovine Fetal Serum (BFS) and Brain Heart Infusion (BHI) broth at 37°C for 24h; growth curves were checked by turbidimetry. Bacterial masses were retrieved during the log and stationary phases and pellets were washed in PBS, weight adjusted and sonicated. Highly hydrophobic molecules (fraction E) were extracted with chloroform-methanol-water (5/10/4, v/v/v); less hydrophobic molecules (fraction F3) were extracted with a solution of butan-1-ol 9%. Fractions were analyzed through a 15% SDS-PAGE system and observed through silver staining and densitometry. Growth was enhanced in BFS during the log phase, without passing through a lag phase; however, the final values of turbidimetry at the stationary phase were lower than in BHI medium. Marked qualitative and quantitative differences in the extracted molecules profile between the growth in BFS and BHI were detected. During the stationary phase, Cp grown in BFS presented only 4 to 5 bands (fractions E and F3, respectively), ranging from 19 kDa to 81 kDa, while Cp grown in BHI presented 11 to 14 molecules, ranging from 13 kDa to 86 kDa. Few bands in the stationary phase extracts were already present in the log phase extracts of Cp grown in both BHI and BFS; particularly, a molecule of 62 kDa found in fraction F3 from BHI in both log and stationary phases was also found in BFS, but 73-fold and 123-fold higher in log and stationary phases, respectively. The expression of few selected cell wall molecules and the higher expression of a specific protein by Cp grown in animal serum might reflect the profile of virulence factors required for infection. Chromatographic purifications of the detected molecules in BFS are being carried out for the development of further studies.