Identification Of The Microbial Factors Contributing To Super-shedding Amongst Isolates Of Shiga Toxin-producing Escherichia Coli O157:H7
Open Access
- Author:
- Katani, Robab
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 10, 2016
- Committee Members:
- Vivek Kapur, Dissertation Advisor/Co-Advisor
Chitrita Debroy, Committee Chair/Co-Chair
Chitrita Debroy, Committee Member
Bhushan M Jayarao, Committee Member
Edward G Dudley, Special Member - Keywords:
- Shiga Toxin-producing Escherichia Coli O157:H7
supershedding
microbial factors
adherence
fimbrial genes
comparative genomic - Abstract:
- Shiga toxin-producing Escherichia coli O157:H7 (O157) are considered as zoonotic foodborne pathogens of major public health concern that cause considerable intestinal and extra-intestinal illnesses in humans. Asymptomatic cattle are the primary reservoir of O157 that are localized in the recto-anal junction (RAJ) of colonized animals and shed in the feces and into the environment. A small subset of cattle, termed supershedders (SS), shed O157 at a rate that is several orders of magnitude (≥ 104 CFU/g of feces) greater than other colonized cattle. Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. To understand microbial factors contributing to supershedding, we sequenced two supershedder O157 isolates, SS17 (GenBank accession no. CP008805) and SS52 (GenBank accession no. CP010304). Comparative genomics analyses identified several polymorphisms in SS17 and SS52 as compared with other previously characterized O157 strains (EC4115, EDL933, Sakai, TW14359), including in a key adherence- and virulence-related loci. Whole genome alignments revealed that both SS strains cluster closely with the lineage I/II spinach outbreak isolates (EC4115 and TW14359) as compared with lineage I outbreak isolates (Sakai and EDL933) or bovine lineage II isolates. Phenotypic analyses revealed a distinctive and strong aggregative locus of enterocyte effacement (LEE)-independent adherence phenotype of wild type SS strains on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. To determine the role of the major fimbrial genes on the adherence of SS strains to host cells, a series of deletion mutants targeting the type 1 fimbriae (fim) and Long Polar Fimbriae (lpf) 1 and 2 operons were constructed in SS strains and EDL933, and the adherence phenotype was evaluated on bovine RSE and human HEp-2 cells. The results provide strong evidence that all three fimbrial operons play a role in the adherence phenotypes of SS strains to bovine RSE and human HEp-2 cells. For instance, the results show that the strain ΔfimHSS17 showed a decrease in the adherence phenotype (from strong aggregative to moderate diffuse) on bovine RSE cells, while the same mutant strain became non-adherent on human HEp-2 cells. In contrast, no change in phenotype was noted with the ΔfimHSS52 and ΔfimHSS77 strains on either RSE or HEp-2 cells. The deletion of the entire fim operon resulted in a decrease in adherence in the SS77 strain on the RSE cells (from strong aggregative to moderate diffuse), yet no change in adherence phenotype in either the SS17 or SS52 strains was observed. Strikingly, Δlpf1 mutants of both tested SS strains (SS17 and SS52) on RSE cells and HEp-2 cells, show a decrease in adherence, suggesting that the lpf1 operon may play a major role in expression of the O157 adherence phenotype to both bovine and human cells. Further, Δlpf1, Δlpf2, double (lpf1/ and lpf2) and triple (fim, lpf1 and lpf2) knockouts in SS isolates and EDL933 provide compelling evidence for a strain and host-cell type dependent role of the fim, lpf1, and lpf2 operons in the adherence phenotype of SS strains of O157 that is likely to be tightly regulated along with other O157 adherence factors. Our comparative genomics and molecular genetic investigations provide key insights on the genomic structure and genetic heterogeneity amongst SS strains of O157, have led to the discovery of a novel adherence phenotype of SS strains to bovine origin RSE cells, provide evidence for a previously un-described role of the fim, lpf1, and lpf2 operons in mediating adherence of SS and other O157 strains to mammalian cells, and highlight opportunities for future investigations into the regulation of adherence in SS strains and the development of potential immunotherapeutic strategies to control O157 super-shedding in cattle, and thereby reduce the risk of transmission, food contamination, and human infection of this major pathogen.