The Pic protease of enteroaggregative Escherichia coli promotes intestinal colonization and growth in the presence of mucin.

Enteroaggregative Escherichia coli (EAEC) is increasingly being recognized as a cause of diarrheal disease in diverse populations. No small animal model is currently available to study this pathogen. We report here that conventional mice orally inoculated with prototype EAEC strain 042 generally became colonized, though the abundance of organisms cultured from their stool varied substantially among individual animals. In contrast, mice whose water contained 5 g/liter streptomycin consistently became colonized at high levels (ca. 10(8) CFU/g of stool). Neither conventional nor streptomycin-treated mice developed clinical signs or histopathologic abnormalities. Using specific mutants in competition with the wild-type strain, we evaluated the contribution of several putative EAEC virulence factors to colonization of streptomycin-treated mice. Our data suggest that the dispersin surface protein and Pic, a serine protease autotransporter secreted by EAEC and Shigella flexneri, promote colonization of the mouse. In contrast, we found no role for the aggregative adherence fimbriae, the transcriptional activator AggR, or the surface factor termed Air (enteroaggregative immunoglobulin repeat protein). To study Pic further, we constructed a single nucleotide mutation in strain 042 which altered only the Pic catalytic serine (strain 042PicS258A). Fractionation of the tissue at 24 h and 3 days demonstrated an approximate 3-log(10) difference between 042 and 042PicS258A in the lumen and mucus layer and adherent to tissue. Strains 042 and 042PicS258A adhered similarly to mouse tissue ex vivo. While no growth differences were observed in a continuous-flow anaerobic intestinal simulator system, the wild-type strain exhibited a growth advantage over 042PicS258A in a culture of cecal mucus and in cecal contents in vitro; this difference was manifest only after 6 h of growth. Moreover, enhanced growth of the wild type was observed in comparison with that of the mutant in minimal medium containing mucin but not in the absence of mucin. The data suggest a novel metabolic role for the Pic mucinase in EAEC colonization.

EilA, a HilA-like regulator in enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a diarrhoeal pathogen in developing and industrialized countries. Most EAEC virulence factors thus far described are encoded on virulence plasmid pAA, yet recent completion of the EAEC genome has suggested the presence of additional factors encoded on chromosomal islands. Previous reports have recognized the presence of a type III secretion system (T3SS), designated ETT2, at the glyU locus of prototype EAEC strain 042, along with possible T3SS effectors at the selC locus. The selC locus was also noted to harbour homologues of Salmonella enterica regulator HilA and of invasin from Yersinia spp., yet previous publications suggested that these loci may be silent. Here, we show that the genes of the selC locus are present inconsistently among a collection of well-characterized EAEC strains. Notably, however, there was perfect correlation between the presence of hilA-homologue eilA and predicted Yersinia invasin homologue gene eaeX. We hypothesized that if expressed, the putative gene product EilA would contribute to EAEC virulence in part by activation of the T3SS and its effectors. An eilA mutant was constructed in EAEC strain 042, and complementation was achieved by cloning the eilA gene under control of an arabinose-dependent promoter. In this system, we observed expression of at least seven genes to be affected by expression of eilA, either directly or indirectly: selC locus genes eipB, eipC, eipD, eicA and eaeX (renamed here air), as well as glyU ETT2 genes eivF and eivA. Notably, the eilA mutant was shown to be less adherent to epithelial cells in culture and to form less abundant biofilms than the isogenic parent. These effects were recapitulated in the air mutant, suggesting that the predicted outer membrane protein product of the air gene is involved as an accessory adhesin and aggregin of EAEC, coexpressed with the T3SS. Our data suggest that the T3SS of EAEC and presumed effectors located on different chromosomal islands may be coordinately activated by EilA, which also activates the genetically linked high molecular weight bacterial surface protein Air. Contributions of this new putative virulence-related regulon in EAEC may include adherence, aggregation, and as yet uncharacterized roles for the T3SS.

Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli.

The gold standard for identification of Enteroaggregative Escherichia coli (EAEC) remains the HEp-2 cell adherence test, which is time-consuming and requires specialized facilities. We evaluated the usefulness of a quantitative biofilm assay to screen for EAEC from a total of 1,042 E. coli strains from children with diarrhea. Bacteria were incubated overnight in high-glucose Dulbecco's modified Eagle's medium using a polystyrene microtiter plate. The plate was stained with crystal violet after washing, and the biofilm was quantified using an enzyme-linked immunosorbent assay plate reader. The aggR gene was evaluated by a polymerase chain reaction. Forty-eight (77.4%) of 62 strains with an optical density at 570 nm (OD(570)) > 0.2 were identified as EAEC by the HEp-2 adherence test, while no EAEC was found in strains with an OD(570) < or = 0.2. Twenty-one aggR+ and 27 aggR - EAEC strains could be screened by an OD(570) > 0.2 using this assay. Although confirmation by a HEp-2 cell adherence test is needed, this biofilm assay is convenient and useful in screening for EAEC.

Use of a continuous-flow anaerobic culture to characterize enteric virulence gene expression.

We developed an in vitro culture method to characterize the expression of bacterial genes under conditions mimicking the colonic environment. Our culture system (the intestinal simulator) comprised a continuous-flow anaerobic culture which was inoculated with fecal samples from healthy volunteers. As a test organism, we employed enteroaggregative Escherichia coli (EAEC), an emerging diarrheal pathogen that is thought to cause infection in both the small and large intestines. After the simulator culture achieved equilibrium conditions, we inoculated the system with prototype EAEC strain 042 and assessed the expression of three EAEC virulence-related genes. We focused particularly on expression of aggR, which encodes a global transcriptional regulator of EAEC virulence factors, and two AggR-regulated genes. By using real-time quantitative reverse transcription-PCR, we showed that aggR expression in the simulator is increased 3- to 10-fold when 042 is grown under low-pH (5.5 to 6.0) conditions, compared with results with neutral pH (7.0). Interestingly, however, this effect was seen only when the strain was grown in the presence of commensal bacteria. We also found that expression of aggR is 10- to 20-fold higher at low NaCl concentrations, and this effect was also observed only in the presence of commensal bacteria. Using coculture and conditioned-media experiments, we identified specific strains of Enterococcus and Clostridium that upregulated aggR expression; in contrast, strains of Lactobacillus and Veillonella downregulated aggR expression. Our data provide new insights into regulation of virulence genes in EAEC and suggest the utility of intestinal simulation cultures in characterizing enteric gene regulation.

Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children.

Diarrhea remains one of the main sources of morbidity and mortality in the world, and a large proportion is caused by diarrheagenic Escherichia coli. In Mongolia, the epidemiology of diarrheagenic E. coli has not been well studied. A total of 238 E. coli strains from children with sporadic diarrhea and 278 E. coli strains from healthy children were examined by PCR for 10 virulence genes: enteropathogenic E. coli (EPEC) eae, tir, and bfpA; enterotoxigenic E. coli (ETEC) lt and st; enteroinvasive E. coli (EIEC) ipaH; enterohemorragic E. coli stx1 and stx2; and enteroaggregative E. coli (EAEC) aggR and astA. EAEC strains without AggR were identified by the HEp-2 cell adherence test. The detection of EAEC, ETEC, EPEC, and EIEC was significantly associated with diarrhea. The incidence of EAEC (15.1%), defined by either a molecular or a phenotypic assay, was higher in the diarrheal group than any other category (0 to 6.0%). The incidence of AggR-positive EAEC in the diarrheal group was significantly higher than in the control group (8.0 versus 1.4%; P = 0.0004), while that of AggR-negative EAEC was not (7.1 versus 4.3%). Nineteen AggR-positive EAEC strains harbored other EAEC virulence genes-aggA, 2 (5.5%); aafA, 4 (11.1%); agg-3a, 5 (13.8%); aap, 8 (22.2%); aatA, 11 (30.5%); capU, 9 (25.0%); pet, 6 (16.6%); and set, 3 (8.3%)-and showed 15 genotypes. EAEC may be an important pathogen of sporadic diarrhea in Mongolian children. Genetic analysis showed the heterogeneity of EAEC but illustrated the importance of the AggR regulon (denoting typical EAEC) as a marker for virulent EAEC strains.

The export of coat protein from enteroaggregative Escherichia coli by a specific ATP-binding cassette transporter system.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen characterized by aggregative adherence (AA) to cultured human mucosal epithelium cells. We have recently characterized a 10.2-kDa protein, called dispersin, which is exported from the bacteria and which promotes dispersal of EAEC across the intestinal mucosa. Here, we present evidence that dispersin is exported by a putative ABC transporter complex, which is encoded by a genetic locus of the EAEC virulence plasmid pAA2. We demonstrate that the locus comprises a cluster of five genes (designated aat-PABCD), including homologs of an inner-membrane permease (AatP), an ATP-binding cassette protein (AatC) and the outer membrane protein TolC (AatA). We show that, like TolC, AatA localizes to the outer membrane independently of its ABC partner. Dispersin appears to require the Aat complex for outer membrane translocation but not for secretion across the inner membrane. We also show that, like the dispersin gene, transcription of the aat cluster is dependent on AggR, a regulator of virulence genes in EAEC. We propose that the aat cluster encodes a specialized ABC transporter, which plays a role in the pathogenesis of EAEC by transporting dispersin out of the bacterial cell.

A novel dispersin protein in enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is a diarrheal pathogen defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture. We have previously shown that EAEC strains secrete a 10-kDa protein that is immunogenic in a human EAEC challenge model. We report here that this protein is encoded by a gene (called aap) lying immediately upstream of that encoding the AggR transcriptional activator, and that aap is under AggR control. The product of aap has a typical signal sequence and is secreted to the extracellular milieu, where it remains noncovalently attached to the surface of the bacterium. EAEC aap mutants aggregate more intensely than the wild-type parent in a number of assays, forming larger aggregates and fewer individual bacteria. Infection of colonic biopsies with wild-type EAEC strain 042 and its aap mutant revealed more dramatic autoagglutination of the mutant compared with the wild-type parent. Our data suggest that the aap gene product participates in formation of a surface coat that acts to disperse the bacteria, thus partially counteracting aggregation mediated by aggregative adherence fimbriae. We have therefore named the aap gene product "dispersin," and we propose that it may be representative of a functional class of colonization factors. Since dispersin is expressed in vivo, is highly immunogenic, and is present in most EAEC strains, it holds considerable promise as an EAEC immunogen.

Regulation of the overlapping pic/set locus in Shigella flexneri and enteroaggregative Escherichia coli.

Most strains of Shigella flexneri 2a and enteroaggregative Escherichia coli carry a highly conserved chromosomal locus which encodes a 109-kDa secreted mucinase (called Pic) and, on the opposite strand in overlapping fashion, an oligomeric enterotoxin called ShET1, encoded by the setA and setB genes. Here, we characterize the genetic regulation of these overlapping genes. Our data suggest that pic and the setBA loci are transcribed as complementary 4-kb mRNA species. The major pic promoter is maximally activated at 37 degrees C in exponential growth phase. Our data suggest that the setB gene is transcribed from a promoter which lies more than 1.5 kb upstream of the setB structural gene; setA may be transcribed via readthrough of the setB transcript and possibly by its own promoter. The long leader of the setB gene provides a strong silencing effect on setB transcription. The signals which provide relief from setB silencing are not clear, but significant induction is observed in a continuous anaerobic culture of human fecal bacteria, suggesting that some complex characteristics of the human intestine act to lift repression of setB expression. Our studies provide the first insights into the mechanisms affecting expression of this unusual virulence locus.