Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli

Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.     

The non-O157 shiga-toxigenic (verocytotoxigenic) Escherichia coli; under-rated pathogens

Following a brief review of the ecology of Escherichia coli in general, the role of Shiga-Toxigenic (Verocytotoxigenic) E. coli (STEC) as pathogens is addressed. While STEC belonging to the serogroup O157 have been extensively studied and shown to be involved in many cases and outbreaks of human disease, the importance of STEC belonging to other serogroups has not been recognized as much. This review addresses the problems associated with these pathogens, demonstrating that increasing the awareness of them is a major part of the problem. This review then demonstrates how widespread isolations especially from food animals and human disease have been, discussing in particular STEC belonging to serogroups O8, O26, O103, O111, O113 and O128. The animal host-specificity of these STEC is also reviewed. In conclusion some methods of improving isolation of these pathogens is addressed.     

Bovine immune response to shiga-toxigenic Escherichia coli O157:H7

Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.     

Iron-Binding Catechols and Virulence in Escherichia coli

Previous work suggested that virulent bacteria, which can grow rapidly in serum, must possess a specific mechanism for removing iron from its transferrin complex. Two strains of Escherichia coli were examined with this in mind. Strain O141, which showed inoculum-dependent growth in serum and multiplied in the mouse peritoneum, secreted iron-binding catechols into both synthetic medium and serum. One of these compounds has an association constant for iron similar to that of transferrin. Both transferrin and ethylenediamine-di-o-hydroxyphenyl acetic acid (EDDA), which have very high affinities for ferric iron, induced catechol synthesis in growing cultures of strain O111. This organism was inhibited by normal horse serum. Further work showed that traces of specific antibody inhibited catechol synthesis by O111 exposed to EDDA; therefore, the existence of this inhibitory process means that the organism can no longer obtain Fe³⁺, which all remains bound to transferrin in serum. In vivo, the inhibition of O111 is similar to that produced by serum in vitro. Neither phagocytosis nor killing by complement appeared to be of any significance during the first 4 h of the infections. Significantly, the purified catechol was capable of abolishing bacteriostasis in vivo. Since these results show that the production of iron-binding catechols is essential for rapid bacterial growth both in vitro and in vivo, these compounds should therefore be considered as true virulence factors. Conversely, any interference by the host with the production or activity of these compounds would constitute an important aspect of antibacterial defense.     

Virulence regulons of enterotoxigenic Escherichia coli

Immunologic Research - Enterotoxigenic Escherichia coli is frequently associated with travelers’ diarrhea and is a leading cause of infant and childhood mortality in developing countries....     

Virulence factors of septicemic Escherichia coli strains

Extraintestinal pathogenic Escherichia coli strains (ExPEC) are the cause of a diverse spectrum of invasive human and animal infections, often leading to septicemia. This review deals with the virulence genes of septicemic ExPEC strains. We discuss the meaning of a virulence gene and survey the genomic, genetic and physiological studies on these strains. Apparently, there are a few virulence factors, which are conserved in the septicemic strains, implying that they are essential for the infection. For the other virulence-related genes a high level of diversity is observed, demonstrating that all stages of the infection can be mediated by a number of alternative virulence factors. The variable profile of virulence genes in septicemic E. coli strains, as well as a prevalence of mobility-related sequences point out the existence of a "mix and match" combinatorial system.     

Virulence properties of asymptomatic bacteriuria Escherichia coli

In asymptomatic bacteriuria (ABU), bacteria colonize the urinary tract without provoking symptoms. Here, we compared the virulence properties of a collection of ABU Escherichia coli strains to cystitis and pyelonephritis strains. Specific urinary tract infection (UTI)-associated virulence genes, hemagglutination characteristics, siderophore production, hemolysis, biofilm formation, and the ability of strains to adhere to and induce cytokine responses in epithelial cells were analyzed. ABU strains were phylogenetically related to strains that cause symptomatic UTI. However, the virulence properties of the ABU strains were variable and dependent on a combination of genotypic and phenotypic factors. Most ABU strains adhered poorly to epithelial cells; however, we also identified a subgroup of strongly adherent strains that were unable to stimulate an epithelial cell IL-6 cytokine response. Poor immune activation may represent one mechanism whereby ABU E. coli evade immune detection after the establishment of bacteriuria.     

Virulence factors in Escherichia coli urinary tract infection.

Uropathogenic strains of Escherichia coli are characterized by the expression of distinctive bacterial properties, products, or structures referred to as virulence factors because they help the organism overcome host defenses and colonize or invade the urinary tract. Virulence factors of recognized importance in the pathogenesis of urinary tract infection (UTI) include adhesins (P fimbriae, certain other mannose-resistant adhesins, and type 1 fimbriae), the aerobactin system, hemolysin, K capsule, and resistance to serum killing. This review summarizes the virtual explosion of information regarding the epidemiology, biochemistry, mechanisms of action, and genetic basis of these urovirulence factors that has occurred in the past decade and identifies areas in need of further study. Virulence factor expression is more common among certain genetically related groups of E. coli which constitute virulent clones within the larger E. coli population. In general, the more virulence factors a strain expresses, the more severe an infection it is able to cause. Certain virulence factors specifically favor the development of pyelonephritis, others favor cystitis, and others favor asymptomatic bacteriuria. The currently defined virulence factors clearly contribute to the virulence of wild-type strains but are usually insufficient in themselves to transform an avirulent organism into a pathogen, demonstrating that other as-yet-undefined virulence properties await discovery. Virulence factor testing is a useful epidemiological and research tool but as yet has no defined clinical role. Immunological and biochemical anti-virulence factor interventions are effective in animal models of UTI and hold promise for the prevention of UTI in humans.     

Virulence of Escherichia coli in ascending urinary-tract infection in mice

The virulence of Escherichia coli strains in ascending urinary-tract infection was studied in mice drinking a 5% glucose solution; factors determining the virulence were examined. Of 33 strains, 8 (group I) infected the bladder and kidney, 10 (group II) infected only the bladder, while the remaining 15 strains (group III) did not cause infection. The adherence of group-I and group-II strains to bladder epithelial cells in vitro was inhibited by D-mannose. In group III, 13 strains barely adhered to the epithelial cells, while two strains showed an adherence unaffected by D-mannose. Most strains in groups I and II agglutinated erythrocytes of guinea-pig, chicken, and horse, and cells of Candida albicans in a mannose-sensitive manner. All strains in groups I and II had fimbriae. Virulence for the urinary tract was not directly related with O-serotype, intraperitoneal virulence, ability to grow in mouse urine, ability to ferment dulcitol, production of haemolysin, susceptibility to serum bactericidal activity, or susceptibility to antibiotics. These results suggest that the adherence of the E. coli to mouse-bladder epithelial cells in a mannose-sensitive manner plays an important role in the development of urinary-tract infection in mice and that the adherence is probably mediated by type-1 or closely related fimbriae.     

Virulence Plasmid Harbored by Uropathogenic Escherichia coli Functions in Acute Stages of Pathogenesis

Urinary tract infections (UTIs), the majority of which are caused by uropathogenic Escherichia coli (UPEC), afflict nearly 60% of women within their lifetimes. Studies in mice and humans have revealed that UPEC strains undergo a complex pathogenesis cycle that involves both the formation of intracellular bacterial communities (IBC) and the colonization of extracellular niches. Despite the commonality of the UPEC pathogenesis cycle, no specific urovirulence genetic profile has been determined; this is likely due to the fluid nature of the UPEC genome as the result of horizontal gene transfer and numerous genes of unknown function. UTI89 has a large extrachromosomal element termed pUTI89 with many characteristics of UPEC pathogenicity islands and that likely arose due to horizontal gene transfer. The pUTI89 plasmid has characteristics of both F plasmids and other known virulence plasmids. We sought to determine whether pUTI89 is important for virulence. Both in vitro and in vivo assays were used to examine the function of pUTI89 using plasmid-cured UTI89. No differences were observed between UTI89 and plasmid-cured UTI89 based on growth, type 1 pilus expression, or biofilm formation. However, in a mouse model of UTI, a significant decrease in bacterial invasion, CFU and IBC formation of the pUTI89-cured strain was observed at early time points postinfection compared to the wild type. Through directed deletions of specific operons on pUTI89, the cjr operon was partially implicated in this observed defect. Our findings implicate pUTI89 in the early aspects of infection.Urinary tract infections (UTIs) represent, by number, one of the most important bacterial infectious diseases in highly industrialized countries (20). Sixty-percent of all women will have at least one UTI within their lifetime (21, 57, 63). This infection results in nearly 7 million physician office visits and $3.5 billion dollars annually in the United States alone (19). It is thought that acute UTIs develop when bacteria from the fecal flora colonize the vaginal and periurethral mucosa and are subsequently introduced into the bladder by urethral ascension. Women who present with an initial episode of acute UTI have a 25 to 44% chance of developing a second and a 3% chance of experiencing three episodes within 6 months of the initial UTI (20). Recurrence occurs despite appropriate antibiotic treatment and clearance of the initial infection from the urine. A large percentage of recurrent UTI are caused by the same strain of bacteria as the initial infection (65). The high frequency of same-strain recurrences supports the notion that a UPEC reservoir may exist in the affected individual.Uropathogenic Escherichia coli (UPEC) is the leading causative agent of UTI, responsible for up to 85% of community-acquired UTI and 25% of nosocomial UTI (62). Using a well-characterized clinical UPEC isolate, UTI89, in a mouse cystitis model, it has been demonstrated that the pathogenesis of UTI involves intracellular and extracellular components (34, 35, 47). The bladder surface is covered with a urothelium composed of very large superficial umbrella cells which are coated with uroplakins that expose a terminal mannose moiety (77). Type 1 pili and its adhesin, FimH, are required for attachment and invasion, thus playing a critical role in the infection process (47, 50, 76). The FimH protein has a negatively charged pocket that accommodates a mannose unit, namely, those exposed by uroplakins on the bladder surface (28). In addition to mannose, it was shown that FimH binds α3β1 integrin subunits expressed by bladder epithelial cells mediating uptake of UPEC (18). Integrins link extracellular matrix proteins with the actin cytoskeleton and can initiate a signaling cascade resulting in bacterial internalization (46, 47). Invasion of UPEC also involves cellular lipid raft components, including uroplakin 1a, caveolin-1, and Rac-1, and internalized bacteria are retained within compartments resembling fusiform vesicles (6, 17). After invasion, bladder epithelial cells are capable of expelling UPEC, presumably as part of an innate defense (69). Another potent innate defense is the exfoliation of umbrella cells and influx of neutrophils (50). A mechanism used by UPEC to combat these defenses is to escape into the cytoplasm of superficial umbrella cells, where they are capable of rapidly replicating into biofilm-like intracellular bacterial communities (IBCs) comprised of 10⁴ to 10⁵ bacteria (1, 34). Upon IBC maturation, bacteria detach from the IBC biomass, flux out of the host cell into the bladder lumen, and spread to neighboring cells forming next-generation IBCs (34, 38). Thus, IBC formation represents a mechanism by which invasion of a single bacterium can result in rapid expansion of UPEC numbers in the urinary tract leading to disease (1, 51). Upon resolution of infection, the terminally differentiated umbrella cells are replaced through Bmp4-specific signaling, restoring the impermeable uroplakin barrier (53). In addition, bacteria can invade the transitional epithelium beneath the umbrella cells, establish a quiescent intracellular reservoir (QIR) and remain dormant until they reemerge and potentially cause a recurrence (52). Reemergence from a QIR could partially explain why recurrent UTI is often caused by a strain identical to the primary infection (65). Recent work has shown that this is a common pathway among UPEC isolates (22), and evidence of this pathway also has been observed in humans (64). The regulatory mechanisms governing IBC development and dispersal in response to environmental cues are still unknown.Despite many similarities among UPEC isolates, genomic features that are unique to UPEC have not yet been identified. Many studies have identified UPEC as a diverse class of E. coli that have evolved multiple and redundant strategies to colonize the urinary tract (75). These diverse genomes have been acquired through horizontal gene transfer (HGT). HGT generates diversity between bacterial species and, through natural selection, contributes to the evolution of bacterial species (16, 40, 66). In addition to genetic variability, newly acquired DNA can enable adaptation to life in a specialized niche through specific factors. The acquisition of DNA by HGT results in the exchange of large regions of DNA called genomic islands (GIs) between bacteria (31). GIs can be acquired in several ways: (i) inheritance of a plasmid that can remain autonomous or recombine into the chromosome, (ii) integration of a lysogenic phage into the chromosome, and/or (iii) insertion of a linear DNA fragment into the chromosome usually by transposition or recombination (14, 43, 70). Some GIs are referred to as pathogenicity islands (PAIs). PAIs contain large genomic DNA regions often greater than 20 kb that carry at least one virulence gene, are inserted within or near tRNA genes, contain direct repeats and mobility sequences, and can frequently be identified by a differing G+C content relative to the bacterial genome (24). In addition, PAIs often do not represent homogeneous pieces of DNA but can instead be mosaiclike structures generated by a multistep process involving genomic acquisition, loss, and rearrangement (25). Study of UPEC PAIs has revealed that they encode numerous virulence factors, including pili and adhesins, toxins, hemolysins, and iron uptake systems. Interestingly, these same virulence factors can be plasmid or phage encoded on intestinal E. coli isolates. Thus, UPEC PAIs could represent former plasmid-derived sequences. In addition, UPEC PAIs carry many cryptic genes, open reading frames (ORFs) of unrelated and even unknown functions, and pseudogenes. A UPEC isolate, UTI89, carries a large extrachromosomal element with many characteristics of UPEC PAIs. This plasmid, pUTI89, has a high proportion of pseudogenes generated by insertion events, ORFs homologous to proteins of nonplasmid origin, and orthologs of enteroinvasive E. coli (EIEC) proteins found on a large virulence plasmid (12). These features describe a UPEC PAI that has potentially not yet incorporated into the genome, known as a “PAI precursor” (41). pUTI89 is approximately 114 kb and, like plasmid F, contains the tra operon for conjugative transfer, as well as genes associated with plasmid replication and inheritance.Although UPEC strains can cause disease in the urinary tract, they can also exist within the human intestinal tract as part of the normal microbiota. Thus, UPEC strains are distinct from the commensal E. coli residing in the gastrointestinal tract in that they possess virulence factors enabling successful transition to and colonization of the urinary tract (8). Factors required for successful infection in addition to type 1 pili are being extensively investigated (76). While studies have demonstrated that additional adhesin systems, toxins, autotransporters, iron acquisition systems, and other factors (reviewed in reference 75) may be important in establishing infection, there is also an abundance of unknown genes labeled as hypothetical or assigned putative functions that may play a role in virulence (32). Here, we studied the role of pUTI89 in the virulence of UPEC. Regions of pUTI89 were shown to be present in the majority of a panel of clinical isolates, suggesting maintenance and transmission within the community. The effect of curing pUTI89 from strain UTI89 was investigated. We found that pUTI89 is involved in an early feature of in vivo infection, even though we could not detect any alterations in growth, type 1 pilus expression and function, or biofilm formation in vitro in the absence of pUTI89. A region on pUTI89 encoding the cjr operon found in EIEC was implicated in this early defect.     

Relationship Between Colicin V Activity and Virulence in Escherichia coli

Colicin V activity is not essential to ColV plasmid-mediated virulence enhancement in Escherichia coli.     

Virulence of enteropathogenic Escherichia coli,a global pathogen

Enteropathogenic Escherichia coli (EPEC) remains an important cause of diarrheal disease worldwide. Research into EPEC is intense and provides a good virulence model of other E. coli infections as well as other pathogenic bacteria. Although the virulence mechanisms are now better understood, they are extremely complex and much remains to be learnt. The pathogenesis of EPEC depends on the formation of an ultrastructural lesion in which the bacteria make intimate contact with the host apical enterocyte membrane. The formation of this lesion is a consequence of the ability of EPEC to adhere in a localized manner to the host cell, aided by bundle-forming pili. Tyrosine phosphorylation and signal transduction events occur within the host cell at the lesion site, leading to a disruption of the host cell mechanisms and, consequently, to diarrhea. These result from the action of highly regulated EPEC secreted proteins which are released via a type III secretion system, many genes of which are located within a pathogenicity island known as the locus of enterocyte effacement. Over the last few years, dramatic increases in our knowledge of EPEC virulence have taken place. This review therefore aims to provide a broad overview of and update to the virulence aspects of EPEC.     

Effect of zinc in enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5′-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5′-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5′-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5′-nucleotidase in vitro than the nucleotide analog α,β-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than α,β-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5′-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host.     

Virulence of Escherichia coli in experimental hematogenous pyelonephritis in mice.

Differences in nephropathogenicity between Escherichia coli strains were studied by following the kinetics of the viable count in the mouse kidney during 8 h after intravenous injection. Assuming as a reference point that at zero time 0.1% of the inoculum was lodged in the kidney, we found that strains fell into three main groups with different behavior patterns: in group I, the viable count fell and remained low; in group II, the viable count first fell and then rose after 4 h, reaching the level of the reference point within 8 h; in group III, the viable count rose rapidly and remained high. The kinetics of the viable counts were also studied in blood, spleen, and liver: group I and II strains behaved similarly; only in the kidney did group II strains show higher counts than group I strains. These data suggest that group II strains are specifically virulent for the mouse kidney. Group III strains also gave higher viable counts in blood and spleen but comparable counts in the liver, suggesting that group III strains are more generally virulent. Fifty percent lethal dose measurements confirmed the conclusion that group I strains are avirulent and group III strains are the most virulent. Possible relationships between behavior pattern and serotype are discussed.     

The Link between Phylogeny and Virulence in Escherichia coli Extraintestinal Infection

Previous studies suggesting a link between Escherichia coli phylogenetic groups and extraintestinal virulence have been hampered by the difficulty in establishing the intrinsic virulence of a bacterial strain. Indeed, unidentified virulence factors do exist, and the susceptibility of the host to infection is highly variable. To overcome these difficulties, we have developed a mouse model of extraintestinal virulence to test the virulence of the strains under normalized conditions. We then assessed the phylogenetic relationships compared to the E. coli reference (ECOR) collection, the presence of several known virulence determinants, and the lethality to mice of 82 human adult E. coli strains isolated from normal feces and during the course of extraintestinal infections. Commensal strains belong mainly to phylogenetic groups A and B1, are devoid of virulence determinants, and do not kill the mice. Strains exhibiting the same characteristics as the commensal strains can be isolated under pathogenic conditions, thus indicating the role of host-dependent factors, such as susceptibility linked to underlying disease, in the development of infection. Some strains of phylogenetic groups A, B1, and D are able to kill the mice, their virulence being most often correlated with the presence of virulence determinants. Lastly, strains of the B2 phylogenetic group represent a divergent lineage of highly virulent strains which kill the mice at high frequency and possess the highest level of virulence determinants. The observed link between virulence and phylogeny could correspond to the necessity of virulence determinants in a genetic background that is adequate for the emergence of a virulent clone, an expression of the interdependency of pathogenicity and metabolic activities in pathogenic bacteria.     

Fitness,Stress Resistance,and Extraintestinal Virulence in Escherichia coli

The extraintestinal virulence of Escherichia coli is dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuring in vitro individual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenic E. coli strains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescent E. coli strain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H₂O₂ resistance, and rpoS sequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains.     

Enhanced CXC chemokine responses of human colonic epithelial cells to locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli

There is increasing evidence that by facilitating translocation of Shiga toxin (Stx) across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leukocytes (PMNs) play a key role in the pathogenesis of Shiga-toxigenic Escherichia coli (STEC) disease. Plasma levels of PMN-attracting CXC chemokines such as interleukin-8 (IL-8) also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. Accordingly, we attempted to determine which STEC factors are responsible for CXC chemokine induction in human colonic epithelial cells. Infection of Hct-8 cells with locus for enterocyte effacement (LEE)-negative STEC strains isolated from patients with severe STEC disease resulted in up-regulation of IL-8, macrophage inflammatory protein 2alpha (MIP-2alpha), MIP-2beta, and ENA-78 mRNA significantly higher and earlier than that elicited by several LEE-positive STEC strains, including the O157:H7 strain EDL933. Similarly, levels of IL-8 protein in LEE-negative STEC-infected Hct-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. The difference in responses could not be attributed to the expression or nonexpression of LEE genes, the presence or absence of an STEC megaplasmid, or differences in O serogroups or in the type or amount of Stx produced. Interestingly, however, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of Hct-8 cells with isolated H21 flagellin elicited IL-8 and MIP-2alpha responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the fliC gene, but not the stx(2) gene, largely abolished the capacity of O113:H21 LEE-negative STEC strain 98NK2 to elicit IL-8 and MIP-2alpha responses in Hct-8 cells. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses seen in cells infected with certain STEC strains are largely attributable to the production of flagellin.