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

We reported earlier that a single gene, tsh, isolated from a strain of avia n pathogenic Escherichia coli (APEC) was sufficient to confer on E. coil K- 12 a hemagglutinin-positive phenotype and that the deduced sequence of the Tsh protein shared homology to the serine-type immunoglobulin A (IgA) prote ases of Neisseria gonorrhoeae and Haemophilus influenzae, In this report we show that E. coil K-12 containing the recombinant tsh gene produced two pr oteins, a 106-kDa extracellular protein and a 33-kDa outer membrane protein , and was also able to agglutinate chicken erythrocytes. N-terminal sequenc e data indicated that the 106-kDa protein, designated Tsh(s), was derived f rom the N-terminal end of Tsh after the removal of a 52-amino-acid N-termin al signal peptide, while the 33-kDa protein, designated Tsh(beta), was deri ved from the C-terminal end of Tsh starting at residue N-1101. The Tsh(s) d omain contains the 7-amino-acid serine protease motif that includes the act ive-site serine (S-259), found also in the secreted domains of the IgA prot eases. However, site-directed mutagenesis of S-259 did not abolish the hema gglutinin activity or the extracellular secretion of Tsh(s) indicating that host-directed proteolysis was mediating the release of Tsh(s). Studies wit h 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 th e IgA protease family, which also includes EspC of enteropathogenic E. coil , EspP of enterohemorragic E. coli, and SepA and VirG of Shigella flexneri, which seem to involve a host endopeptidase to achieve extracellular releas e of their N-terminal domains. In proteolytic studies conducted in vitro, T sh(s) did not cleave the substrate of the IgA proteases, human IgA1 or chic ken IgA, and did not show proteolytic activity in a casein-based assay, Cor relation of Tsh expression and hemagglutination activity appears to be a ve ry complex phenomenon, influenced by strain and environmental conditions. N evertheless, for both APEC and recombinant E. coil K-12 strains containing the tsh gene, it was only the whole bacterial cells and not the cell-free s upernatants that could confer hemagglutinin activity. Our results provide i nsights into the expression, secretion, and proteolytic features of the Tsh protein, which belongs to the growing family of gram-negative bacterial ex tracellular virulence factors, named autotransporters, which utilize a self -mediated mechanism to achieve export across the bacterial cell envelope.