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

Abstract

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 Tsh(s), 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(β), was derived from the C-terminal end of Tsh starting at residue N1101. The Tsh(s) 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 Tsh(s) indicating that hostdirected proteolysis was mediating the release of Tsh(s). Studies with an E. coli K-12 ompT mutant strain showed that the surface protease OmpT was not needed for the secretion of Tsh(s). 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, Tsh(s) 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.