Resumo

Previous studies of our laboratory have demonstrated that Corynebacterium pseudotuberculosis (Cp) up-regulates the expression of some selected structural/membrane proteins when grown in animal serum. In the present work, the aim was to investigate the immunogenic profile of the up-regulated structural proteins expressed by Cp grown in animal serum in comparison to commercial medium. A high virulence Cp strain (VD57) was grown in bovine fetal serum (BFS) and brain heart infusion (BHI) broth at 37°C with shaking for 24h in triplicate, until reaching the stationary phase; the bacterial pellets were retrieved and washed in PBS, adjusted to the same weight and sonicated. A delipidation step was performed for three times with chloroform-methanol-water (5/10/4, v/v/v), and the membrane proteins were extracted for three times with butanol 9% in water. All the protein extracts of all replicates were pooled together and analyzed through a 15% SDS-PAGE system and observed through silver staining and densitometry; proteins were also transferred to nitrocellulose membranes for carrying out an immunoblotting with serum from infected sheep. Qualitative and quantitative differences in the profile of extracted molecules between the growth in BFS and BHI were detected. The protein profile obtained in BHI presented 20 visible bands, ranging from 18 kDa to 66 kDa, while Cp grown in BFS presented 8 bands, ranging from 19 kDa to 66 kDa. Based on densitometry, the molecule with 54 kDa presented an expression rate (BFS:BHI) of 8.01, while a band of 31 kDa presented the ratio of 41.16, confirming the up-regulation of some proteins of Cp grown in animal serum. It was detected by Immunobloting that Cp cultured in BHI presented 5 immunoreactive bands, while Cp grown in BFS presented 7 immunoreactive antigens. The immunodominant antigen was the 31 kDa protein for both protein extracts, presenting similar densitometric peaks in the electrophoretogram. Particularly, the immunobloting profile of Cp grown in BFS presented three new reactive antigens, with estimated molecular weight of 65 kDa, 48 kDa and 24 kDa, which were not expressed by Cp grown in BHI. Based on the obtained results, it can be concluded that the expression of some virulence factors is dependant of contact between Cp and host factors such as the serum and/or blood environment.