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Chronological set of E. coli O157:H7 bovine strains establishes a role for repeat sequences and mobile genetic elements in genome diversification.

ABSTRACT: BACKGROUND:Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant foodborne pathogen that resides asymptomatically within cattle and other ruminants. The EHEC genome harbors an extensive collection of mobile genetic elements (MGE), including multiple prophage, prophage-like elements, plasmids, and insertion sequence (IS) elements. RESULTS:A chronological collection of EHEC strains (FRIK804, FRIK1275, and FRIK1625) isolated from a Wisconsin dairy farm (farm X) comprised a closely related clade genetically differentiated by structural alterations to the chromosome. Comparison of the FRIK804 genome with a reference EHEC strain Sakai found a unique prophage like element (PLE, indel 1) and an inversion (1.15?Mb) situated symmetrically with respect to the terminus region. Detailed analysis determined the inversion was due to homologous recombination between repeat sequences in prophage. The three farm X strains were distinguished by the presence or absence of indel 3 (61 kbp) and indel 4 (48 kbp); FRIK804 contained both of these regions, FRIK1275 lacked indel 4, and indels 3 and 4 were both absent in FRIK1625. Indel 3 was the stx2 prophage and indel 4 involved a deletion between two adjacent prophage with shared repeat sequences. Both FRIK804 and FRIK1275 produced functional phage while FRIK1625 did not, which is consistent with indel 3. Due to their involvement in recombination events, direct and inverted repeat sequences were identified, and their locations mapped to the chromosome. FRIK804 had a greater number and overall length of repeat sequences than E. coli K12 strain MG1655. Repeat sequences were most commonly associated with MGE. CONCLUSIONS:This research demonstrated that three EHEC strains from a Wisconsin dairy farm were closely related and distinguished by variability within prophage regions and other MGE. Chromosome alterations were associated with recombination events between repeat sequences. An inventory of direct and inverted repeat sequences found a greater abundance and total length of repeat sequences in the EHEC strains compared to E. coli strain MG1655. The locations of the repeat sequences were biased towards MGE. The findings from this study expand our understanding of the precise molecular events and elements that contributed to genetic diversification of wild-type EHEC in the bovine and farm environments.

SUBMITTER: Stanton E

PROVIDER: S-EPMC7430833 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Chronological set of E. coli O157:H7 bovine strains establishes a role for repeat sequences and mobile genetic elements in genome diversification.

Stanton Eliot E Wahlig Taylor A TA Park Dongjin D Kaspar Charles W CW

BMC genomics 20200817 1

<h4>Background</h4>Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant foodborne pathogen that resides asymptomatically within cattle and other ruminants. The EHEC genome harbors an extensive collection of mobile genetic elements (MGE), including multiple prophage, prophage-like elements, plasmids, and insertion sequence (IS) elements.<h4>Results</h4>A chronological collection of EHEC strains (FRIK804, FRIK1275, and FRIK1625) isolated from a Wisconsin dairy farm (farm X) comprised ...[more]

PMID: 32807088

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Project description:The bovine gastrointestinal (GI) tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients preferentially used by EHEC in the bovine intestine would help to develop ecological strategies to reduce EHEC carriage. However, the carbon sources that support the growth of EHEC in the bovine intestine are poorly documented. In this study, a very low concentration of glucose, the most abundant monomer included in the cattle dietary polysaccharides, was detected in bovine small intestine contents (BSIC) collected from healthy cows at the slaughterhouse. Six carbohydrates reported to be included in the mucus layer covering the enterocytes [galactose, N-acetyl-glucosamine (GlcNAc), N-acetyl- galactosamine (GalNAc), fucose, mannose and N-acetyl neuraminic acid (Neu5Ac)] have been quantified for the first time in BSIC and accounted for a total concentration of 4.2?mM carbohydrates. The genes required for enzymatic degradation of the six mucus-derived carbohydrates are highly expressed during the exponential growth of the EHEC strain O157:H7 EDL933 in BSIC and are more strongly induced in EHEC than in bovine commensal E.?coli. In addition, EDL933 consumed the free monosaccharides present in the BSIC more rapidly than the resident microbiota and commensal E.?coli, indicating a competitive ability of EHEC to catabolize mucus-derived carbohydrates in the bovine gut. Mutations of EDL933 genes required for the catabolism of each of these sugars have been constructed, and growth competitions of the mutants with the wild-type strain clearly demonstrated that mannose, GlcNAc, Neu5Ac and galactose catabolism confers a high competitive growth advantage to EHEC in BSIC and probably represents an ecological niche for EHEC strains in the bovine small intestine. The utilization of these mucus-derived monosaccharides by EDL933 is apparently required for rapid growth of EHEC in BSIC, and for maintaining a competitive growth rate as compared with that of commensal E.?coli. The results suggest a strategy for O157:H7 E.?coli survival in the bovine intestine, whereby EHEC rapidly consumes mucus-derived carbohydrates that are poorly consumed by bacteria belonging to the resident intestinal microbiota, including commensal E.?coli.

Project description:BackgroundEnterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in bovine faecal samples at slaughter. Cattle do not show clinical symptoms upon infection, but for humans the consequences after consuming contaminated beef can be severe. The immune response against EHEC in cattle cannot always clear the infection as persistent colonization and shedding in infected animals over a period of months often occurs. In previous infection trials, we observed a primary immune response after infection which was unable to protect cattle from re-infection. These results may reflect a suppression of certain immune pathways, making cattle more prone to persistent colonization after re-infection. To test this, RNA-Seq was used for transcriptome analysis of recto-anal junction tissue and ileal Peyer's patches in nine Holstein-Friesian calves in response to a primary and secondary Escherichia coli O157:H7 infection with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served as controls.ResultsIn tissue of the recto-anal junction, only 15 genes were found to be significantly affected by a first infection compared to 1159 genes in the ileal Peyer's patches. Whereas, re-infection significantly changed the expression of 10 and 17 genes in the recto-anal junction tissue and the Peyer's patches, respectively. A significant downregulation of 69 immunostimulatory genes and a significant upregulation of seven immune suppressing genes was observed.ConclusionsAlthough the recto-anal junction is a major site of colonization, this area does not seem to be modulated upon infection to the same extent as ileal Peyer's patches as the changes in gene expression were remarkably higher in the ileal Peyer's patches than in the recto-anal junction during a primary but not a secondary infection. We can conclude that the main effect on the transcriptome was immunosuppression by E. coli O157:H7 (Stx-) due to an upregulation of immune suppressive effects (7/12 genes) or a downregulation of immunostimulatory effects (69/94 genes) in the ileal Peyer's patches. These data might indicate that a primary infection promotes a re-infection with EHEC by suppressing the immune function.

Project description:BackgroundThe cattle gastrointestinal tract (GIT) is the main enterohemorrhagic Escherichia coli (EHEC) reservoir. In order to identify nutrients required for the survival or multiplication of EHEC in the bovine GIT, we compared the transcriptomes of the EHEC O157:H7 reference strain EDL933 cultured in vitro in bovine digestive contents (DCs) (rumen, small intestine and rectum) using RNA-sequencing.ResultsGene expression profiles showed that EHEC EDL933 activated common but also specific metabolic pathways to survive in the different bovine DCs. Mucus-derived carbohydrates seem important in EHEC nutrition in posterior DCs (small intestine and rectum) but not in rumen content. Additional carbohydrates (xylose, ribose, mannitol, galactitol) as well as gluconeogenic substrates (aspartate, serine, glycerol) would also be used by EHEC as carbon and/or nitrogen sources all along the bovine GIT including the rumen. However, xylose, GalNac, ribose and fucose transport and/or assimilation encoding genes were over-expressed during incubation in rectum content compared with rumen and intestine contents, and genes coding for maltose transport were only induced in rectum. This suggests a role for these carbohydrates in the colonization of the cattle rectum, considered as the major site for EHEC multiplication. In contrast, the transcription of the genes associated with the assimilation of ethanolamine, an important nitrogen source for EHEC, was poorly induced in EHEC growing in rectum content, suggesting that ethanolamine is mainly assimilated in the cattle rumen and small intestine. Respiratory flexibility would also be required for EHEC survival because of the redundancy of dehydrogenases and reductases simultaneously induced in the bovine DCs, probably in response to the availability of electron donors and acceptors.ConclusionEHEC EDL933 showed a high flexibility in the activation of genes involved in respiratory pathways and assimilation of carbon and nitrogen sources, most of them from animal origin. This may allow the bacterium to adapt and survive in the various bovine GIT compartments. Obtaining a better understanding of EHEC physiology in bovine GIT is a key step to ultimately propose strategies to limit EHEC carriage and shedding by cattle.

Project description:Enterohaemorrhagic Escherichia coli (EHEC) are responsible for outbreaks of food- and water-borne illness. The bovine gastrointestinal tract (GIT) is thought to be the principle reservoir of EHEC. Knowledge of the nutrients essential for EHEC growth and survival in the bovine intestine may help in developing strategies to limit their shedding in bovine faeces thus reducing the risk of human illnesses. To identify specific metabolic pathways induced in the animal GIT, the transcriptome profiles of EHEC O157:H7 EDL933 during incubation in bovine small intestine contents (BSIC) and minimal medium supplemented with glucose were compared. The transcriptome analysis revealed that genes responsible for the assimilation of ethanolamine, urea, agmatine and amino acids (Asp, Thr, Gly, Ser and Trp) were strongly up-regulated suggesting that these compounds are the main nitrogen sources for EHEC in BSIC. A central role for the gluconeogenesis pathway and assimilation of gluconeogenic substrates was also pinpointed in EHEC incubated in BSIC. Our results suggested that three amino acids (Asp, Ser and Trp), glycerol, glycerol 3-phosphate, L-lactate and C4-dicarboxylates are important carbon sources for EHEC in BSIC. The ability to use gluconeogenic substrates as nitrogen sources (amino acids) and/or carbon sources (amino acids, glycerol and lactate) may provide a growth advantage to the bacteria in intestinal fluids. Accordingly, aspartate (2.4 mM), serine (1.9 mM), glycerol (5.8 mM) and lactate (3.6 mM) were present in BSIC and may represent the main gluconeogenic substrates potentially used by EHEC. A double mutant of E. coli EDL933 defective for phosphoenolpyruvate synthase (PpsA) and phosphoenolpyruvate carboxykinase (PckA), unable to utilize tricarboxylic acid (TCA) intermediates was constructed. Growth competition experiments between EHEC EDL933 and the isogenic mutant strain in BSIC clearly showed a significant competitive growth advantage of the wild-type strain further illustrating the importance of the gluconeogenesis pathway in maintaining EHEC in the bovine GIT.

Dataset Information

Chronological set of E. coli O157:H7 bovine strains establishes a role for repeat sequences and mobile genetic elements in genome diversification.

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Chronological set of E. coli O157:H7 bovine strains establishes a role for repeat sequences and mobile genetic elements in genome diversification.

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