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 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 Tshbeta, 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.

FIG. 1

Subcellular localization of two proteins expressed when the tsh gene is present in CC118. Bacterial strains were grown on CFA agar at 26°C for 48 h. Subcellular fractions were isolated as described in Materials and Methods. Aliquots were resolved by SDS–10% PAGE. (A) Gel stained with Coomassie brilliant blue. (B) Gel transferred to nitrocellulose and Western immunoblotted with absorbed anti-Tsh antibody. The two proteins produced by CC118(pYA3107) that are not produced by CC118(pACYC184) are marked by arrows. Lanes 1 to 5, CC118(pYA3107); lanes 6 to 10, CC118(pACYC184). Lanes 1 and 6, whole cells; lanes 2 and 7, soluble fractions; lanes 3 and 8, inner membrane fractions; lanes 4 and 9, outer membrane fractions; lanes 5 and 10, supernatant fractions. The molecular mass markers (in kilodaltons) are shown to the left of panel A.

FIG. 2

Expression of the 106- and 33-kDa proteins by tsh mutants. CC118 containing two tsh mutations derived by transposon mutagenesis (43) and CC118 containing the wild-type tsh gene were grown on CFA agar at 26°C for 48 h. Supernatants and outer membrane proteins were isolated and resolved by SDS–10% PAGE and stained with Coomassie brilliant blue. The two proteins produced by CC118(pYA3107) that are not produced by CC118 containing the tsh mutations are marked by arrows. Lanes 1 to 3, supernatant fractions; lanes 4 to 6, outer membrane proteins. Lanes 1 and 4, CC118(pYA3315); lanes 2 and 5, CC118(pYA3321); lanes 3 and 6, CC118(pYA3107). The molecular mass markers (in kilodaltons) are shown on the left.

FIG. 3

Immunoblot of supernatant fractions from χ7122, χ7141, and CC118(pYA3107). Bacterial strains were grown on CFA agar as described in Materials and Methods. Lane 1, χ7141, a tsh mutant derived from χ7122 (43); lane 2, CC118(pYA3107); lane 3, χ7122. The molecular mass markers (in kilodaltons) are shown on the left.

FIG. 4

Immunoblot of LE392 and BL21 expressing Tsh and Tsh-T259. Bacterial strains were grown on CFA agar at 26°C for 48 h or at 37°C for 24 h. Hemagglutination activity was determined as described in Materials and Methods. Supernatants were isolated, acetone precipitated and resolved by SDS–10% PAGE as described in Materials and Methods. After transfer to nitrocellulose the 106-kDa protein was detected with absorbed anti-Tsh antibody. Lanes 1 to 3, LE392 grown at 26°C; lanes 4 to 6, LE392 grown at 37°C; lanes 7 to 9, BL21 grown at 26°C. Lanes 1, 4, and 7 show strains transformed with pBluescript II(SK); lanes 2, 5, and 8 show strains transformed with pYA3108. Lanes 3, 6, and 9 show strains transformed with pYA3287. Molecular mass markers (in kilodaltons) are shown to the left of the figure. Hemagglutination titers for each strain are shown under the figure.

FIG. 5

Immunoblots of human IgA1 (A) or chicken IgA (B). Bacterial strains were grown as described in Materials and Methods. Supernatants were isolated and added to aliquots of human IgA1 or chicken IgA. After 14 h of incubation at 37°C, the entire mixture was resolved by SDS–10% PAGE and then transferred to nitrocellulose. This membrane was immunoblotted with anti-human IgA1 α-chain antibody (in panel A) or anti-chicken IgA α-chain antibody (in panel B). The molecular mass markers (in kilodaltons) are shown on the right. (A) Lane 1, human IgA1 alone; lane 2, H. influenzae N187; lane 3, CC118(pACYC184); lane 4, CC118(pYA3107); lane 5, χ7141; lane 6, χ7122. (B) Lane 1, chicken IgA1 alone; lane 2, CC118(pACYC184); lane 3, CC118(pYA3107); lane 4, χ7141; lane 5, χ7122.

FIG. 6

Casein-based assay for proteolytic activity. L broth supernatants were collected as described in Materials and Methods. Samples were incubated at 37°C for 16 h, and proteolytic activity was measured with a fluorometer. Averages from three independent experiments are shown; each assay was done in triplicate. Cultures of XL1-Blue(pWKS30) and XL1-Blue(pYA3418) were induced with 1 mM IPTG for 1 h at an optical density at 600 nm of 0.6. The samples tested are indicated by different columns as follows: A, L broth medium; B, 5 μg of trypsin; C, 5 μg of Hap_s; D, supernatant from culture of χ7122; E, supernatant from culture of χ7141; F, supernatant from culture of XL1-Blue(pWKS30); G, supernatant from culture of XL1-Blue(pYA3418); H, supernatant from culture of H. influenzae DB117(pJS106); F, supernatant from culture of H. influenzae DB117(pGJB103).

FIG. 7

Immunoblot of supernatant fractions from χ7122, χ7141, and CC118(pYA3108) grown under different conditions. Bacterial strains were grown on CFA agar at 26°C for 48 h or at higher temperatures for 24 h. CFA agar was supplemented with 0.15 M NaCl as indicated. Hemagglutination activity was determined as described in Materials and Methods. Lane 1, χ7141 grown at 26°C; lane 2, χ7122, grown at 26°C; lane 3, χ7122 grown at 37°C; lane 4, χ7122 grown at 42°C; lane 5, χ7122 grown at 26°C on CFA supplemented with 0.15 M NaCl; lane 6, CC118(pYA3108) grown at 26°C, lane 7, CC118(pYA3108) grown at 37°C; lane 8, CC118(pYA3108) grown at 42°C. Molecular mass markers (in kilodaltons) are shown to the left of the figure. Hemagglutination titers for each strain are shown under the figure.