Characterization of the avian pathogenic Escherichia coli hemagglutinin Tsh, a member of the immunoglobulin A protease-type family of autotransporters

• Published in: Infection and immunity Vol. 67; no. 2; pp. 772 - 781
• Main Authors: Stathopoulos, C, Provence, D.L, Curtiss, R. III
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.02.1999
• Subjects: Adhesins, Escherichia coli - genetics; Adhesins, Escherichia coli - metabolism; Animals; bacterial proteins; Bacteriology; binding proteins; Biological and medical sciences; Birds; Carrier Proteins - genetics; Carrier Proteins - metabolism; chickens; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Hemagglutination Tests; Hemagglutinins - genetics; Hemagglutinins - metabolism; Immunoglobulin A; Microbiology; Molecular and Cellular Pathogenesis; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; proteinases; Rabbits; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Shigella flexneri; Serine endopeptidases; Enzyme; Hemagglutinin; Secretion; Escherichia coli; Glycoprotein; Host agent relation; Gene expression; IgA-specific serine endopeptidase; Characterization; Pathogenicity; Peptidases; Vertebrata; Enzymatic activity; Pathogen strain; Bacteria; Hydrolases; Veterinary; Aves; Enterobacteriaceae
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/IAI.67.2.772-781.1999

We reported earlier that a single gene, tsh, isolated from a strain of avian pathogenic Escherichia coli (APEC) was sufficient to confer on E. coli K-12 a hemagglutinin-positive phenotype and that the deduced sequence of the Tsh protein shared homology to the serine-type immunoglobulin A (IgA) proteases of Neisseria gonorrhoeae and Haemophilus influenzae. In this report we show that E. coli K-12 containing the recombinant tsh gene produced two proteins, a 106-kDa extracellular protein and a 33-kDa outer membrane protein, and was also able to agglutinate chicken erythrocytes. N-terminal sequence data indicated that the 106-kDa protein, designated Tshs, was derived from the N-terminal end of Tsh after the removal of a 52-amino-acid N-terminal signal peptide, while the 33-kDa protein, designated Tsh beta, was derived from the C-terminal end of Tsh starting at residue N1101. The Tshs domain contains the 7-amino-acid serine protease motif that includes the active-site serine (S259), found also in the secreted domains of the IgA proteases. However, site-directed mutagenesis of S259 did not abolish the hemagglutinin activity or the extracellular secretion of Tshs indicating that host-directed proteolysis was mediating the release of Tshs. Studies with an E. coli K-12 ompT mutant strain showed that the surface protease OmpT was not needed for the secretion of Tshs. Tsh belongs to a subclass of the IgA protease family, which also includes EspC of enteropathogenic E. coli, EspP of enterohemorragic E. coli, and SepA and VirG of Shigella flexneri, which seem to involve a host endopeptidase to achieve extracellular release of their N-terminal domains. In proteolytic studies conducted in vitro, Tshs did not cleave the substrate of the IgA proteases, human IgA1 or chicken IgA, and did not show proteolytic activity in a casein-based assay. Correlation of Tsh expression and hemagglutination activity appears to be a very complex phenomenon, influenced by strain and environmental conditions. Nevertheless, for both APEC and recombinant E. coli K-12 strains containing the tsh gene, it was only the whole bacterial cells and not the cell-free supernatants that could confer hemagglutinin activity. Our results provide insights into the expression, secretion, and proteolytic features of the Tsh protein, which belongs to the growing family of gram-negative bacterial extracellular virulence factors, named autotransporters, which utilize a self-mediated mechanism to achieve export across the bacterial cell envelope.