The Mycobacterium avium subsp. paratuberculosis 35 kDa protein plays a role in invasion of bovine epithelial cells

• Published in: Microbiology (Society for General Microbiology) Vol. 149; no. 8; pp. 2061 - 2069
• Main Authors: Bannantine, John P, Huntley, Jason F. J, Miltner, Elizabeth, Stabel, Judith R, Bermudez, Luiz E
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.08.2003; Society for General Microbiology
• Subjects: Animals; Antibodies, Bacterial - biosynthesis; Antigens, Bacterial - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Bacterial Proteins - physiology; Bacteriology; Base Sequence; Biological and medical sciences; Cattle; Cattle Diseases - microbiology; Cell Line; Cloning, Molecular; DNA, Bacterial - genetics; Epithelial Cells - microbiology; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Intestines - microbiology; MBP-MMP protein; Microbiology; Molecular Weight; Mycobacterium avium; Mycobacterium avium subsp. paratuberculosis - genetics; Mycobacterium avium subsp. paratuberculosis - immunology; Mycobacterium avium subsp. paratuberculosis - pathogenicity; Mycobacterium avium subsp. paratuberculosis - physiology; Paratuberculosis - microbiology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Virulence - physiology; Membrane protein; Bovine; Digestive system; Paratuberculosis; Gut; Mycobacterium avium; Mycobacterial infection; Infection; Vertebrata; Mammalia; Mycobacteriales; Pathogenic; Bacteriosis; Mycobacteriaceae; Bacteria; Epithelial cell; Actinomycetes; Artiodactyla; Veterinary; Ungulata
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.26323-0

1 National Animal Disease Center, USDA-ARS, 2300 North Dayton Avenue, Ames, IA 50010, USA 2 Iowa State University, Department of Veterinary Pathology, Ames, IA, USA 3 Oregon State University, Department of Biomedical Sciences, Corvallis, OR, USA Correspondence John P. Bannantine jbannant{at}nadc.ars.usda.gov Mycobacterium avium subsp. paratuberculosis ( M. paratuberculosis ) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. This study showed that a gene encoding the M. paratuberculosis 35 kDa major membrane protein (MMP) is expressed 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 Escherichia 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 %. In similar invasion experiments with M. paratuberculosis incubated in low oxygen tension, these antibodies and protein decreased invasion by 60 %. Collectively, these data show that the 35 kDa MMP is a surface exposed protein that plays a role in invasion of epithelial cells. The authors suggest that the MMP is a virulence factor of M. paratuberculosis that may be important in the initiation of infection in vivo . Abbreviations: MBP, maltose-binding protein; MDBK, Madin–Darby bovine kidney; MMP, major membrane protein