Submitted to: Infection and Immunity
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/28/2003
Publication Date: 8/1/2003
Citation: Bannantine, J.P., Huntley, J.F., Miltner, E., Stabel, J.R., Bermudez, L.E. 2003. The mycobacterium avium supspecies paratuberculosis 35 kda protein plays a role in invasion of bovine epithelial cells. Infection and Immunity. Interpretive Summary: The mechanism of how Mycobacterium paratuberculosis causes disease in cattle is poorly understood. While it is generally accepted that this bacterium can survive within bovine macrophages, which are immune cells designed to kill most bacteria, the method it uses to attached and colonize the bovine intestine is unknown. This communication describes a protein produced by M. paratuberculosis that plays a role in invasion of bovine intestinal cells. The protein has been termed major membrane protein (MMP) because we show that it is present on the surface of the bacterium. The experiments that revealed the protein's role in invasion are called competitive exclusion experiments. In these experiments, when you add the purified protein, along with M. paratuberculosis, you observe a decrease in the number of bacteria that invade (infect) the epithelial cells relative to that seen when only M. paratuberculosis is added to cells. These studies reveal the first M. paratuberculosis protein that plays a role in the initial interactions with bovine epithelial cells. Results from these studies may have implications for vaccine development.
Technical Abstract: Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. In this study, we observed that a gene encoding the M. paratuberculosis 35-kDa major membrane protein (MMP) is transcribed at a higher level in low oxygen and high osmolarity conditions that are similar to the environment of the intestine. In addition, cattle with Johne's disease produced antibodies against MMP, suggesting that the protein is present during infection. The gene encoding MMP was cloned and expressed as a fusion protein with the maltose binding protein (MBP/MMP) in E. coli. Rabbit antisera were raised against a M. paratuberculosis whole cell sonicate and MMP-specific antibodies were purified from these sera by affinity chromatography. MMP was localized to the surface of M. paratuberculosis by immunoelectron microscopy and by immunoblot analysis of fractionated protein lysates. Both anti-MMP antibodies and MBP/MMP protein inhibited M. paratuberculosis invasion of cultured Madin-Darby bovine kidney cells by 30%. Similar invasion experiments with M. paratuberculosis incubated in low oxygen tension, decreased invasion by 60%. Collectively, these data show that the 35-kDa MMP protein is a surface exposed protein that plays a role in invasion of epithelial cells. From these studies, we suggest that the major membrane protein is a virulence factor of M. paratuberculosis that may be important in the initiation of infection in vivo.