Although Shiga toxin is the definitive virulence factor of STEC, the capacity to compete with commensal flora and colonize the human intestinal epithelium is critical for pathogenesis. The mechanism by which LEE-positive STEC strains adhere intimately to the enterocyte surface has been studied extensively, whereas the mechanism(s) by which LEE-negative STEC strains adhere to the intestinal mucosa is less well understood. Nevertheless, the importance of adhesins is reflected in the broad range of such proteins discovered in LEE-positive as well as in LEE-negative STEC strains, including Saa, (26
), Lpf (10
), EibG (18
), Iha (38
), Efa1 (23
), Cah (41
), ECP (30
), HCP (47
), and EhaA (45
). Nearly all LEE-positive as well as many LEE-negative STEC strains carry megaplasmids, which encode putative accessory virulence factors, including some of the above-mentioned proteins. Significantly, the megaplasmids of highly virulent LEE-negative STEC strains such as O113:H21 strains EH41 and 98NK2 are much larger than those of the classical LEE-positive STEC O157:H7 strains and encode a broad range of additional virulence factors, many of which appear to be unique to LEE-negative strains (22
In this study, we have characterized one such putative virulence factor, the AT Sab, which is present in some but not all LEE-negative STEC serotypes/groups including multiple isolates of O113:H21 and single isolates of O23 and O82:H8. In both 98NK2 and EH41, the sab gene is located on megaplasmid pO113, approximately 1.3 kb downstream of the hemolysin locus ehx. All sab-positive STEC strains tested were also positive for ehxA as well as the autoagglutinating adhesin gene saa and the subtilase cytotoxin gene subA; the latter genes are located 20 and 28 kb, respectively, downstream of sab on pO113. However, the absence of sab in several other LEE-negative STEC strains that carry one or more of the other megaplasmid-borne genes tested (ehxA, saa, or subA) underscores the heterogeneity of STEC strains with respect to their complement of megaplasmid-encoded accessory virulence factors.
Western blot analysis indicated that the level of expression of the Sab protein in wild-type 98NK2 is low under the in vitro conditions employed in this study (growth in LB medium or DMEM). Indeed, it was detectable only when concentrated cell lysates were examined. It is possible that Sab is expressed at high levels in vivo, although we found no difference in sab gene expression levels by real-time reverse transcription-PCR when cells were grown in LB medium or DMEM in the presence or absence of HEp-2 cells (data not shown). Nevertheless, the introduction of sab on a high-copy-number plasmid in either the JM109 or 98NK2sab::kan background resulted in much higher levels of expression. SDS-PAGE and Western blotting results suggested that the protein is capable of forming multimers. Moreover, Sab was present exclusively in the outer membrane protein fraction and was exposed on the surface of intact E. coli cells, as judged by the accessibility to exogenous antibody using immunofluorescence or ELISA.
The surface localization of Sab is consistent with our observation that it confers the capacity to adhere diffusely to HEp-2 cells when expressed in JM109 cells. Moreover, the purified fluorescently labeled passenger domain of Sab bound to the surface of HEp-2 cells, suggesting that Sab may act as a direct adhesin rather than as an indirect facilitator of adherence. Sab also appears to be largely responsible for the capacity of wild-type 98NK2 cells to form a biofilm on polystyrene surfaces. In contrast, biofilm formation was negligible in 98NK2sab::kan as well as in the poorly adherent STEC food isolate MW10, which lacks an intact sab gene. Furthermore, the biofilm formation defect in 98NK2sab::kan was complemented by transformation with pBsab but not with empty vector. Likewise, biofilm formation by JM109(pBsab) was significantly greater than that by JM109(pB).
The C terminus of Sab shares homology with the prototypic trimeric ATs Hia and YadA, and given its mobility on SDS-PAGE gels, which is indicative of multimerization, its location in the outer membrane, its exposure on the E. coli
surface, and its unequivocal role in adherence to epithelial cells and biofilm formation, we hypothesize that Sab is also a member of the trimeric AT family. Several other ATs were previously reported to promote biofilm formation, for example, Cah (41
), EhaA (45
), UpaG (42
), Ag43 (8
), and AIDA (34
). The capacity to form biofilms may enhance the survival of pathogens in different environmental niches, such as in food products or in the gastrointestinal tracts of humans or animal reservoirs of infection. It is particularly noteworthy that even the low baseline level of sab
expression in wild-type 98NK2 was sufficient to confer a substantial level of biofilm formation, and this was completely abolished in the otherwise isogenic mutant 98NK2sab
. Other studies have shown that the trimeric AT proteins YadA, NhhA, BadA, and UpaG promote binding to extracellular matrix proteins, such as fibronectin, laminin, or collagen (31
). However, we found no evidence that either purified Sab protein or E. coli
JM109 carrying sab
was capable of binding to any of these extracellular matrix proteins (result not shown). Some ATs have also been shown to mediate autoagglutination, but this was not observed for Sab (result not shown).
In summary, we have shown that the STEC megaplasmid-encoded putative trimeric AT family protein Sab promotes adherence to human epithelial cells and mediates biofilm formation. The distribution of sab
, although based on an analysis of a relatively small number of clinical and environmental STEC isolates, indicates that Sab, like the autoaggregative adhesin Saa (26
), is associated with LEE-negative STEC strains. Thus, both adhesins may contribute to intestinal colonization in STEC strains that lack the capacity to form A/E lesions on enterocytes. It is also noteworthy that apart from encoding Sab and Saa, as well as additional putative accessory virulence factors including subtilase cytotoxin (SubAB) (25
) and two additional SPATE family secreted serine protease ATs, EspP (4
) and EpeA (16
), the megaplasmids of highly virulent STEC O113:H21 strains 98NK2 and EH41 are self-transmissible (36
). Thus, the assembly of such a diverse payload of virulence factors onto a single mobile DNA element is likely to have contributed to the evolution of hypervirulent LEE-negative STEC strains.