Heterologous Expression of a Gene Encoding a 35 kDa Protein of Mycobacterium avium paratuberculosis in Escherichia coli

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.

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Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2. Properties of the recombinant protein

Biochimie ◽

10.1016/s0300-9084(97)83518-3 ◽

1997 ◽

Vol 79 (5) ◽

pp. 303-308 ◽

Cited By ~ 7

Author(s):

E. De Vendittis ◽

M.R. Amatruda ◽

G. Raimo ◽

V. Bocchini

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Heterologous Expression ◽

Elongation Factor ◽

Gene Encoding ◽

Expression In Escherichia Coli ◽

Elongation Factor 2

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Molecular and Immunological Analyses of the Mycobacterium avium Homolog of the Immunodominant Mycobacterium leprae 35-Kilodalton Protein

Infection and Immunity ◽

10.1128/iai.66.6.2684-2690.1998 ◽

1998 ◽

Vol 66 (6) ◽

pp. 2684-2690 ◽

Cited By ~ 22

Author(s):

James A. Triccas ◽

Nathalie Winter ◽

Paul W. Roche ◽

Andrea Gilpin ◽

Kathleen E. Kendrick ◽

...

Keyword(s):

Amino Acid ◽

Mycobacterium Avium ◽

Amino Acid Level ◽

Streptomyces Griseus ◽

Amino Acid Identity ◽

Delayed Type Hypersensitivity ◽

Gene Encoding ◽

Genes Encoding ◽

Species Specific ◽

35 Kda Protein

ABSTRACT The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare,M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.

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