Sab, a novel autotransporter of locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21, contributes to adherence and biofilm formation

Abstract

Shiga-toxigenic Escherichia coli (STEC) strains cause serious gastrointestinal disease, which can lead to potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the production of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm formation). The 4,296-bp sab gene encodes a 1,431-amino-acid protein with the features of members of the AT protein family. When expressed in E. coli JM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2) cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sab deletion mutant was also defective in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a naturally occurring deletion in sab was similarly defective in biofilm formation. PCR analysis indicated that sab is present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.

FIG. 1.

Genetic organization of the sab gene and domain structure of the Sab protein. (A) The sab locus is flanked on one side by LH0146 (encoding a putative OmpA family lipoprotein) and the ehx locus (encoding STEC hemolysin) and on the other side by LH0148 (unknown function). The region shown corresponds to nucleotides 114000 to 124000 of the pO113 sequence from STEC EH41 (GenBank accession number NC_007365). (B) The domain structure of the Sab protein (aa 1 to 1431) was predicted using SignalP (3), Pfam (2), and NCBI Protein BLAST (20).

FIG. 2.

Western blot analysis using anti-Sab. Strains were grown in LB medium (A and C) or DMEM (B) at 37°C, and lysates or protein extracts were prepared and separated by SDS-PAGE, blotted, and probed with anti-Sab, as described in Materials and Methods. (A) Whole-cell lysates. Lanes: 1, 98NK2sab::kan(pB); 2, 98NK2sab::kan(pBsab); 3, JM109(pB); 4, JM109(pBsab). (B) French press lysates. Lanes: 1, 98NK2; 2, 1:10 dilution of 98NK2; 3, 98NK2sab::kan. (C) Inner and outer membrane fractions. Lanes: 1, 98NK2; 2, 98NK2sab::kan. The arrow indicates the mobility of the 160-kDa Sab species.

FIG. 3.

Immunofluorescence microscopy. Log-phase cultures of 98NK2sab::kan(pBsab) (A) and 98NK2sab::kan(pB) (B) were formalin fixed and labeled with anti-Sab followed by Alexa 594-conjugated donkey anti-mouse secondary antibody (see Materials and Methods). The fluorescent image for each strain is accompanied by the phase-contrast image for the corresponding field.

FIG. 4.

Surface expression of Sab on whole unfixed cells judged by use of ELISA. Cells from log-phase cultures of 98NK2, 98NK2sab::kan, and 98NK2sab::kan(pBsab) were probed with anti-Sab and secondary goat anti-mouse immunoglobulin G-alkaline phosphatase antibody, as described in Materials and Methods. Surface expression was quantitated by measuring the A₄₀₅. Data are means ± standard deviations of data from three independent experiments (**, P < 0.01; ***, P < 0.005 by Student's unpaired, two-tailed t test [relative to 98NK2sab::kan]).

FIG. 5.

Adherence assays. (A) Quantitative adherence of JM109(pBsab) and JM109(pB) to HEp-2 and Henle 407 cells (see Materials and Methods). Data correspond to means ± standard deviations of data from quadruplicate assays from two independent experiments. (*, P < 0.05 by Student's unpaired, two-tailed t test). (B) Giemsa staining of adherent bacteria on HEp-2 cell monolayers visualized by light microscopy.

FIG. 6.

Protein-binding assay. HEp-2 monolayers were exposed to 0 μg/ml (A) or 20 μg/ml (B) Oregon Green-labeled Sab protein for 2.5 h at 37°C. Unbound protein was removed by washing, and fixed cells were viewed by confocal microscopy.

FIG. 7.

Biofilm formation on polystyrene plates. The indicated strains were grown in 96-well polystyrene plates for 18 h. Plates were then washed and stained with crystal violet (see Materials and Methods). Biofilm formation was quantitated by measuring the A₅₇₀ after solubilization of the stained bacteria. Data are means ± standard deviations (n = 24) (***, P < 0.005 by Student's unpaired, two-tailed t test [relative to the respective negative control]).