Structural signature of Ser83Leu and Asp87Asn mutations in DNA gyrase from enterotoxigenic Escherichia coli and impact on quinolone resistance.

Enterotoxigenic Escherichia coli (ETEC) is among the most frequent microorganisms causing traveler's diarrhea (TD). Quinolones are potent antimicrobial agents used for the treatment of TD. Resistance to quinolones is typically caused by substitutions in QRDR region of gyrA subunit of DNA gyrase. The aim of this study was to seek insights into the effect of these substitutions at structural level and their association with observed quinolone resistance. Majority of the ETEC strains have gyrA mutations at amino acid position 83 and 87. To understand the quinolone resistance mechanism at molecular level, we have studied the interaction of wild type and mutant forms of ETEC gyrA with nalidixic acid and ciprofloxacin by molecular modeling using Discovery Studio and LeadIt. All the mutants had reduced affinity towards both ciprofloxacin and nalidixic acid relative to the wild type due to the mutations introduced in gyrA. Besides Ser83 and Asp87, for nalidixic acid binding Arg91 and His45 residues were observed to be critical while in ciprofloxacin binding Lys42 and Arg91 residues played a significant role. Amino acid substitutions contribute to the emergence of drug resistance in sensitive strains by causing structural alterations leading to reduced affinity of the drug towards receptor. Analysis of the effect of amino acid substitutions at structural level is of utmost importance to establish possible associations between mutations and the diseases. These studies accelerate the identification of pharmaceutical targets for relevant treatments and could also be helpful in guiding the design of further experimental research.

EatA, an immunogenic protective antigen of enterotoxigenic Escherichia coli, degrades intestinal mucin.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality due to infectious diarrhea in developing countries for which there is presently no effective vaccine. A central challenge in ETEC vaccinology has been the identification of conserved surface antigens to formulate a broadly protective vaccine. Here, we demonstrate that EatA, an immunogenic secreted serine protease of ETEC, contributes to virulence by degrading MUC2, the major protein present in the small intestinal mucous layer, and that removal of this barrier in vitro accelerates toxin access to the enterocyte surface. In addition, we demonstrate that vaccination with the recombinant secreted passenger domain of EatA (rEatAp) elicits high titers of antibody and is protective against intestinal infection with ETEC. These findings may have significant implications for development of both subunit and live-attenuated vaccines against ETEC and other enteric pathogens, including Shigella flexneri, that express similar proteins.

Enterotoxigenic Escherichia coli secretes a highly conserved mucin-degrading metalloprotease to effectively engage intestinal epithelial cells.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of death due to diarrheal illness among young children in developing countries, and there is currently no effective vaccine. Many elements of ETEC pathogenesis are still poorly defined. Here we demonstrate that YghJ, a secreted ETEC antigen identified in immunoproteomic studies using convalescent patient sera, is required for efficient access to small intestinal enterocytes and for the optimal delivery of heat-labile toxin (LT). Furthermore, YghJ is a highly conserved metalloprotease that influences intestinal colonization of ETEC by degrading the major mucins in the small intestine, MUC2 and MUC3. Genes encoding YghJ and its cognate type II secretion system (T2SS), which also secretes LT, are highly conserved in ETEC and exist in other enteric pathogens, including other diarrheagenic E. coli and Vibrio cholerae bacteria, suggesting that this mucin-degrading enzyme may represent a shared virulence feature of these important pathogens.

Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence.

Biofilm formation and binding specificities of CFA/I, CFA/II and CS2 adhesions of Enterotoxigenic Escherichia coli and CfaE-R181A mutant

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/ CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF'lacIq/∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF'lacIq/pEU5881) showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.

The prevalence of enterotoxigenic E. coli isolated from the stools of children aged 0-10 years with diarrhea in mid-anatolia region, Turkey

The stool samples from 245 patients with diarrhea were tested for heat labile toxin (LT) and heat stable toxins (ST) by passive latex agglutination and enzyme immunoassay methods respectively. Twelve (4.9 percent) enterotoxigenic E. Coli ETEC strains were isolated. Five strains (2 percent) expressed ST, and 7 (2.8 percent) expressed LT.