FIG. 5.

Effect of the oxidoreductase mutation on presentation of proteins in the cell surface. MDBK cells were infected at 37°C with wt M. avium subsp. paratuberculosis or with the ΔOx mutant. Extracellular bacteria were collected after 1 h of infection and surface-labeled using biotinamidohexanoic acid N-hydroxysuccinimide ester (20 mg/ml). Labeled bacteria were lysed, and 0.5-μg portions of the cell lysates were analyzed on a 12% SDS-PAGE gel. Gels were transferred to nitrocellulose membranes and probed with HRP-conjugated streptavidin. Lane 2 shows a biotinylated protein identified in extracellular wt M. avium subsp. paratuberculosis upon contact with MDBK cells that was not present in the ΔOx mutant (lane 4). wt M. avium subsp. paratuberculosis and the ΔOx mutant incubated in HBSS for 1 h at 37°C were used as controls (lanes 1 and 3).