Anaerobic conditions promote expression of Sfp fimbriae and adherence of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM to human intestinal epithelial cells

Abstract

The sfp gene cluster, unique to sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM strains, encodes fimbriae that mediate mannose-resistant hemagglutination in laboratory E. coli strains but are not expressed in wild-type SF EHEC O157:NM strains under standard laboratory conditions. We investigated whether Sfp fimbriae are expressed under conditions that mimic the intestinal environment and whether they contribute to the adherence of SF EHEC O157:NM strains to human intestinal epithelial cells. The transcription of sfpA (encoding the major fimbrial subunit) was upregulated in all strains investigated, and all expressed SfpA and possessed fimbriae that reacted with an anti-SfpA antibody when the strains were grown on solid media under anaerobic conditions. Sfp expression was absent under aerobic conditions and in liquid media. Sfp upregulation under anaerobic conditions was significantly higher on blood agar and a medium simulating the colonic environment than on a medium simulating the ileal environment (P < 0.05). The induction of Sfp fimbriae in SF E. coli O157:NM strains correlates with increased adherence to Caco-2 and HCT-8 cells. Our data indicate that the expression of Sfp fimbriae in SF E. coli O157:NM strains is induced under conditions resembling those of the natural site of infection and that Sfp fimbriae may contribute to the adherence of the organisms to human intestinal epithelium.

FIG. 1.

Influence of anaerobiosis on sfpA transcription in SF E. coli O157:NM strains. Strains were passaged four times on the media indicated under aerobic or anaerobic (Anaerocult A) conditions, and sfpA mRNA was quantified using RT-PCR. *, sfpA transcription under anaerobic conditions was significantly higher than that under aerobic conditions on blood agar for strains 493/89 (P = 0.015), 3072/96 (P = 0.038), and E03/71 (P = 0.038). **, sfpA transcription under anaerobic conditions was significantly higher than that under aerobic conditions on SCEM for strains 493/89 (P = 0.020) and 3072/96 (P = 0.038). The upregulation of sfpA mRNA was significantly higher on blood agar and SCEM than on SIEM for strain 493/89 (P, 0.020 and 0.038, respectively) and significantly higher on blood agar than on SIEM for strain 3072/96 (P = 0.038). All data are means from three independent experiments. Error bars, standard deviations.

FIG. 2.

Visualization of Sfp fimbriae in HB101/pSFO157-E11 and in wild-type SF EHEC O157:NM strain 493/89 using electron microscopy after negative staining (A) and immunogold staining with an anti-SfpA antibody (B). (Panels 1) HB101/pSFO157-E11; (panels 2) vector control strain HB101/pBluescript II KS(+); (panels 3) strain 493/89 grown under anaerobic conditions; (panels 4) strain 493/89 grown under aerobic conditions. Bars represent 500 nm (A, panels 1 to 4, and B, panels 2 and 3) or 150 nm (B, panels 1 and 4).

FIG. 3.

Adherence of SF EHEC O157:NM strain 493/89 grown aerobically (panels 3) or anaerobically (panels 4) to HCT-8 (A) and Caco-2 (B) cells compared with the adherence of HB101/pSFO157-E11 (panels 2) and the vector control strain HB101/pBluescript II KS(+) (panels 1). Bars represent 10 μm.