Diversity of Shiga toxin-converting phage and CRISPR loci in Shiga toxin-producing Escherichia coli: Evolutionary and pathogenicity implications.

摘要

Shiga toxin-producing Escherichia coli (STEC) are a group of foodborne pathogens, including the notorious serotype O157:H7 and the six most frequently observed non-O157 serogroups in the United States, known as the "big six". Cattle are the major reservoir. Both O157:H7 and the big-six are under strict regulation in beef; the regulations are known as the zero tolerance policy. The diseases caused by STECs vary in severity, ranging from mild diarrhea, bloody diarrhea, to a life threatening condition called hemolytic uremic syndrome (HUS). Although the susceptibility of the human host plays a large role in determining severity, genetic differences between STEC can greatly influence the virulence as well. The genomic diversity of STECs is mainly driven by horizontally acquired genomic regions, within which most virulence factors are encoded. Studying the diversity of the virulence-associated horizontally acquired regions can enhance our understanding of STEC pathogenesis. In addition, depending on the level of diversity, certain genomic regions may be used as detection and subtyping markers for pathogen prevention, outbreak control and epidemiological investigation. Therefore, the overall goal of the present research was to characterize the diversity of two genomic regions in STECs and study their implications on pathogenesis, strain evolution and utility for subtyping.;One virulence factor, Shiga toxin (Stx), is encoded within a temperate prophage region on the E. coli O157:H7 genome. The production of Stx is crucial for HUS development. More than 200 O157:H7 strains were sequenced in a project assessing O157:H7 genomic diversity. However, the important stx-converting prophage regions were not well assembled due to the presence of other prophages with high sequence similarity. In Chapter 3, we explored the stx-converting phage diversity within a subset of this O157:H7 collection by isolating and sequencing total phage DNA. The complete genomes of 22 stx2a-converting phages were recovered. Sequence comparison identified 9 phage sequence types (PSTs), and genome and proteome analysis grouped them into 3 clusters (i.e. PST1, PST2 and PST3). Phage genome diversity was driven by both copies of IS629 and sequence variations within the phage early regulatory gene regions. The PST2 cluster phages were phylogenetically related to phages identified in strains previously associated with high HUS rate. Meanwhile, we also demonstrated, by using 5 strains carrying identical stx2a-converting prophage, that the host genome impacts Stx2 production. Overlaying the phage genome sequence data and the whole bacteria genome phylogeny suggested that stx2a-converting phage of PST2 may have been acquired multiple times independently during the stepwise evolution model from O55:H7 to O157:H7.;Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system in prokaryotes that consists of short DNA repeats separated by sequences called spacers that derive from viruses and other foreign DNA. While CRISPR sequences in E. coli are diverse, the acquisition of new spacers has only been observed in engineered strains, implying the relatively slow evolution of CRISRPs in E. coli and making them potential subtyping markers for phylogenetically related strains. In fact, a CRISPR-based qPCR assay has been previously developed for the big-six and O157 STECs. In Chapter 4, by examining the CRISPR loci in over 500 isolates, we provided more insights into CRISPR diversity in STECs. The general spacer content and order conservation within each serogroup confirmed the specificity of the primers used in the qPCR assay. Spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup provided an explanation for the observed false-negative reactions in the qPCR results. Isolates expressing the same flagellar antigen (i.e. H7, H2 and H11) also shared similar spacer content and order, implying their potential common origin. These isolates serve as a source of false-positives for the qPCR assay. Using a collection of strains belonging to a well-defined stepwise evolutionary path, we provided the evidence that CRISPR evolution in E. coli proceeds via spacer deletion rather than acquisition.;In summary, the present research characterized the genomic diversity of stx2a-converting phage and CRISPRs in STECs, providing insights into the evolution and pathogenesis of this pathogen group, and generated genome data for further mechanistic studies.