Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains remain a formidable cause of diarrheal disease. To identify novel surface proteins of ETEC, we performed TnphoA mutagenesis of prototype ETEC strain H10407 and discovered a secreted protein not previously recognized in ETEC. DNA sequencing of the interrupted locus in mutant TnphoA.977 revealed a candidate 4,095-bp open reading frame without significant homology to commensal E. coli K-12 genomic DNA. Translation of this sequence revealed that it encoded a predicted peptide of 147.7 kDa that bears significant homology to members of the autotransporter family of bacterial virulence factors, particularly the serine protease autotransporters of the Enterobacteriaceae proteins. The gene identified in H10407, eatA (ETEC autotransporter A), encodes a potential serine protease motif (GDSGSP) in the secreted amino-terminal domain, and the predicted peptide shows more than 80% homology with SepA, a virulence protein secreted by Shigella flexneri. DNA hybridization and PCR demonstrated that eatA resides on the 92-kDa pCS1 virulence plasmid of H10407 and that it is present in multiple clinical ETEC strains. Immunoblots with antisera directed against a recombinant EatA passenger protein fragment identified a 110-kDa protein in supernatants purified from H10407 but not from the TnphoA.977 mutant or H10407-P, which lacks pCS1. EatA possesses serine protease activity that is abolished by mutations within a serine protease catalytic triad formed by residues H(134), D(162), and S(267). Finally, interruption of the eatA gene retarded fluid accumulation in the rabbit ileal loop model, suggesting that this autotransporter contributes to the virulence of ETEC.

FIG. 1.

(A) Map of pJTH019 demonstrating the location of the TnphoA insertion within the 5′ end of eatA. BspHI sites (Bs) flank the insert. Maps of plasmids bearing full-length eatA (pSP009) and eatA subclones (pJTH024, pSP007). Relative locations of eatA locus DNA probes 1 and 2 used in DNA hybridization studies are depicted at the bottom of the map. (B) Hybridization of eatA gene probe 1 in Southern blot of BamHI-digested DNA from isolated ETEC H10407 virulence plasmids of 92, 64, and 6 kDa. (C) PCR and colony hybridization (hyb) data (probe 2) from H10407, H10407-P, as well as other clinical ETEC strains from diverse geographic origins. Size markers of 1.6 and 2.0 kb are shown at the left of each gel image.

FIG. 2.

ClustalW alignment (http://www.ch.embnet.org/index.html) of the predicted peptide sequences of EatA and its closest homologue, the Shigella flexneri protein SepA. Regions shaded black indicate identity. The two proteins have several features in common with other SPATE proteins, including an extended signal peptide and a putative serine protease site within the proposed passenger domain. Amino acids which form part of the serine protease catalytic triad are indicated (*).

FIG. 3.

(A) Proposed structure of EatA molecule. Predicted signal peptide is shaded grey. Cleavage site between amino acids 56 and 57 was predicted by Signal P (http://www.cbs.dtu.dk/services/SignalP/) (43). The passenger domain is unshaded and extends from A57 to approximately amino acid 1098, based on prior sequence alignments of SepA and other autotransporter proteins (3). The carboxy-terminal β-barrel transport region is hatched, with predicted Kyte-Doolittle (30) hydrophilicity plot (MacVector). The location of amino acids H¹³⁴, D¹⁶², and S²⁶⁷ within the conserved SPATE GDSGSP motif (amino acids 265 to 270), proposed to form the serine protease catalytic triad, are shown above the passenger domain. The black bar depicts the region of EatA (amino acids 88 to 581) represented in the recombinant protein used to raise polyclonal rabbit antisera. (B) Anti-EatA immunoblot of supernatants from H10407 (wild type), H10407-P (cured of the 92-kDa virulence plasmid), and TnphoA.977, bearing the eatA::TnphoA mutation. (C) Anti-EatA immunoblot of concentrated supernatants from clinical ETEC strains and a recombinant E. coli strain expressing the cloned eatA gene on pSP009.

FIG. 4.

(A) Cleavage of synthetic p-nitroanilide oligopeptide substrates by recombinant EatA protein. (AAPM = MeOSuc-Ala-Ala-Pro-Met-pNA, AAPL = Suc-Ala-Ala-Pro-Leu-pNA, AAPF = Suc-Ala-Ala-Pro-Phe-pNA, AAPV = MeSuc-Ala-Ala-Pro-Val-pNA, MeOH, GGF = Suc-Gly-Gly-Phe-pNA, VPF = Suc-Val-Pro-Phe-pNA, FLF = Suc-Phe-Leu-Phe-pNA). Hydrolysis of each substrate was assessed by monitoring the increase in absorption over time at 405 nm. Activity was then expressed as V_max in milliunits min⁻¹. Values for each peptide represent the average of the V_max data from three independent experiments relative to the values obtained for cleavage of AAPM substrate under the same conditions. (B) Effect of mutations within the putative serine protease catalytic triad or addition of phenylmethylsulfonyl fluoride on EatA enzymatic activity. Values represent activity of each mutant protein on the indicated substrate relative to the parent protein. (C) Mutations in the serine protease catalytic triad have no effect on the secretion of the EatA passenger domain. Immunoblots of concentrated supernatants from LMG194 bearing pBAD-TOPO-based plasmids expressing either the wild-type eatA gene (pSP014) or serine protease mutants (pSP018, pSP020, and pSP019, respectively). Concentrated supernatants from the ΔeatA deletion strain (eatA::Cm) and the deletion strain complemented with either the pBAD-TOPO vector containing a lacZ control insert (pSP013) or pSP014 are shown for comparison.

FIG. 5.

(A) EatA leads to accelerated virulence in a rabbit model of ETEC infections. The graph represents data obtained from six separate rabbit experiments. In each experiment, ETEC H10407 (wild type) was tested against the eatA deletion strain (▵) and uninfected (control) loops. (B) Histopathology of rabbit ileal loops 7 h after inoculation with bacteria. Images (×10 magnification) represent separate loops obtained from the same rabbit.