Resumo

Mycobacterias have specific glycoconjugated antigens in the cell wall that act as immunomodulatory molecules involved in the survival inside phagocytic cells; these molecules interact with macrophages mainly in the early stages of infection, during the acute phase, being recognized by innate immunity receptors. Thus, these antigens have potential applicability in diagnostic tests for the early detection of diseased patients. Since Corynebacterium pseudotuberculosis (Cp) is phylogenetically related to Mycobacterium sp., it is supposed that Cp cell wall also presents such structures. In this study, we investigated the profile of antigenic recognition of Cp cell wall components by sheep IgM antibodies. A low virulent (T1) and a high virulent (VD57) Cp strain were grown in Brain Heart Infusion (BHI) broth for 72h; the bacterial masses were retrieved and the pellets were washed 3x with PBS, and sonicated. A primary extraction was performed with chloroform-methanol-water (5/10/4, v/v/v) for three times; the supernatants were pooled, dried, and subjected to a butan-1-ol–water (1/1, vol/vol) partition. The aqueous phase generated by the butan-1-ol–water partition was designated as F2, which includes lipophosphoglycans. A secondary extraction was carried out with the pellets made with butan-1-ol 9% in water; supernatants were pooled, dried and designated as F3, which includes peptidoglycans. The two fractions of both bacterial strains were analyzed through a 15% SDS-PAGE system and observed through silver staining, or transferred to nitrocellulose membranes for an immunoblot made with sera from experimentally infected sheep, as well as seronegative samples. Differences in the profile of antigenic recognition by IgM in the different fractions were detected, in which fraction F2 presented an antigen with estimated molecular weight of 11 kDa, while fraction F3 presented recognition of the same antigen plus a reactive band with estimated molecular weight of 19 kDa. The intensity of the recognition was higher with T1 strain antigen. These antigens might be associated with Cp virulence mechanisms; chromatographic purifications of these molecules are being carried out for further studies.