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.Shiga-toxigenic Escherichia coli (STEC) strains are prominent food-borne pathogens that cause watery or bloody diarrhea and hemorrhagic colitis, which can progress to the lifethreatening hemolytic-uremic syndrome (HUS) (15,21,29). In order to establish and maintain an infection, STEC strains are equipped with a diverse array of virulence factors. Among these factors, Shiga toxin has been considered to be a sine qua non of virulence, as reviewed previously (21, 29). However, attachment of STEC to the human intestinal mucosa is a critical first step in pathogenesis. Many STEC strains, including those of the highly prevalent O157:H7 serotype, carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the capacity to produce attaching and effacing (A/E) lesions on the intestinal epithelium, similarly to those produced by enteropathogenic E. coli strains (11, 35). These STEC strains are often referred to as enterohemorrhagic E. coli (EHEC), although this classification is ill defined. A/E lesions are characterized by ultrastructural changes including the remodeling of the host cell cytoskeleton and intimate attachment of the bacteria to the cell surface (11,35). The process of the generation of A/E lesions involves the expression of the eae gene, which encodes intimin, an outer membrane surface adhesin, and the delivery of the intimin receptor Tir and several other effector proteins into host cells via the LEE-encoded type III secretion apparatus (reviewed in references 5 and 11).However, many STEC isolates from cases of ...

Incidence and Virulence Determinants of Verocytotoxin-Producing Escherichia coli Infections in the Brussels-Capital Region, Belgium, in 2008–2010

“…Seropathotype E, comprising VTEC that does not cause disease in humans, was not taken into account, since all VTEC strains originated from clinical cases. A recently developed PCR-based method was applied for the identification of the VT1 and VT2 subtypes ( Additional virulence genes eae, ehxA, saa, subA, espP, katP, and etpD were searched for as described previously (7,20,46,47,58). All isolates were screened for the presence of O island 122 (OI-122)-associated genes pagC, sen, nleB, nleE, efa1, and efa2 (30,61).…”

Serotypes and Virulence Profiles of Non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Bovine Farms

“…In addition to the stx genes, other virulence factors encoded by eaeA (intimin), tir (espE) (translocated intimin receptor), espA (EspA protein), and espB (EspB protein) are required for intimate attachment and the formation of the attaching and effacing (A/E) lesions characteristic of STEC infection (10,13,79). Other clinically significant virulence markers include hlyA (pO157 enterohemolysin releasing hemoglobin from red blood cells), katP (pO157 catalase peroxidase that defends the cell against oxidative damage), etpD (pO157, encodes part of a type II secretory pathway transporting proteins across the outer membrane) (73), lpf (chromosomal long polar fimbriae) (72), espP (pO157 extracellular serine protease autotransporter), saa (pO113 STEC agglutinating adhesion) (55), sab (pO113 STEC autotransporter) (33), toxB (pO157 adhesion), and iha (chromosomal iron-regulated gene A homolog adhesin) (68,70).…”

Molecular Mechanisms That Mediate Colonization of Shiga Toxin-Producing Escherichia coli Strains

“…PCR analyses using primers to detect the presence of putative adhesin genes in O113:21 strains with lower (strain MW10) and higher (strain 98NK2) adherence to Henle 407 cells revealed the presence of one open reading frame (ORF) present exclusively in 98NK2. Further characterization indicated that this ORF encodes a 146-kDa protein with features characteristic of an autotransporter protein, which was designated the STEC autotransporter mediating biofilm formation or Sab (39). By using a collection of LEE-positive and LEE-negative STEC strains, researchers found the sab gene in a subset of LEEnegative strains, while no LEE-positive STEC strains were found to possess this gene.…”

“…Analysis of the hypervirulent LEEnegative STEC O113:H21 strain revealed the presence of another autotransporter which was involved in adherence of STEC to abiotic surfaces and epithelial cells (39). PCR analyses using primers to detect the presence of putative adhesin genes in O113:21 strains with lower (strain MW10) and higher (strain 98NK2) adherence to Henle 407 cells revealed the presence of one open reading frame (ORF) present exclusively in 98NK2.…”