Characterization of the novel factor paa involved in the early steps of the adhesion mechanism of attaching and effacing Escherichia coli

Abstract

Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.

FIG. 1.

Light microscopy micrographs of ileal explants inoculated with the wild-type O45 strain 86-1390 (A) or with the LEE-negative strain 862 (B). Strain 86-1390 showed a typical intimate-adherence pattern (arrowheads) with irregularity of the associated epithelial cells, whereas a loose association of bacteria with the intestinal mucosa of some villi with no obvious change in associated epithelial cells (arrow) was observed for negative-control strain 862. Magnification, ×400.

FIG. 2.

Adherence of wild-type strains and their paa mutant strains. (A) paa mutant strain M155 (n = 18) showed a decreased number of intact ileal villi with bacterial adherence to epithelial cells, compared to wild-type PEPEC strain 86-1390 (n = 12) and to the complemented mutant strain M155c (n = 20). The porcine strain 862 (n = 15), which does not possess the LEE, was used as a negative control. (B) paa mutant strain E22Δpaa (n = 19) showed a decreased number of intact ileal villi compared to wild-type REPEC strain E22 (n = 19) and to the complemented mutant strain E22c (n = 10). Error bars, standard deviations of the means. Asterisk, statistically significant difference (P < 0.0001, when compared by Kruskal-Wallis test) from wild-type strains 86-1390 (A) and E22 (B).

FIG. 3.

Transmission electron micrographs of ileal explants inoculated with the wild-type O45 strain 86-1390 (A; magnification, ×20,664), the complemented mutant strain M155c (B; magnification, ×20,702), or TnphoA mutant M155 (C; magnification, ×13,500). Typical A/E lesions were observed for both wild-type and complemented-mutant strains, whereas bacteria in the lumen without any direct contact with the epithelium were observed for the mutant M155.

FIG. 4.

The nucleotide sequence of paa and its flanking sequences and the deduced amino acid sequences. The putative −10 and −35 promoter sites, the ribosome binding site (RBS), and the putative transcription terminator are underlined. The translation initiation codon and the TAG translation termination codon are in boldface. Vertical arrow, potential peptide signal cleavage site; arrowhead, insertion site of TnphoA.

FIG. 5.

Alignment of the deduced amino acid sequences of the Paa of the 86-1390 strain and the Paa proteins of the O157:H7 EDL933 and Sakai E. coli strains, the AcfC protein of V. cholerae, and the PEB3 protein of C. jejuni. ∗, identical or conserved residues in all sequences in the alignment; colons, conserved substitutions; periods, semiconserved substitutions. The amino acid sequence alignment was performed with the Clustal W program.

FIG. 6.

Reduction of the percentage of intact villi showing intimate adherence when pig ileal explants are inoculated with PEPEC strain 86-1390 following treatment with anti-Paa antibodies, compared to percentages for explants inoculated with strain 86-1390 following treatment with antibodies from hens immunized with a sonicate preparation from host strain M15(pREP4) (T −). The porcine strain 862, which does not have the LEE, was used as a negative control. Asterisk, statistically significant difference (P < 0.0001, when compared by Kruskal-Wallis tests) from the T (−) treatment.

FIG. 7.

Transmission electron micrographs showing the uniform distribution of immunogold-labeled Paa protein (arrow) over the bacterial surface of the complemented strain M155c (A) following overnight growth at 37°C in TSB. When anti-Paa serum was adsorbed against the Paa protein, only a few gold beads were observed for strain M155c, mostly in the background (B). Bars = 300 nm.