Although both the cecum and colon had high levels of EHEC, previous studies have found that EHEC closely associates with the cecal epithelium and not the colon in mice Eaton et al. This possibility may account for why a significant difference was observed only in the cecum. Previously, cathepsin B release was shown in EHEC-infected human monocytes but could not be demonstrated using mouse monocytes Cheng et al.
This suggests that other cathepsins may be released or other cell types may release cathepsin B in the mouse during EHEC infection. Thus, both the acute infection phase and the chronic impact of GI inflammation pose risks to human health. Previously, multiple non-LEE effector proteins were found to interfere with innate immune system signaling pathways Gao et al. Thus, while host cells may recognize microbial factors and activate inflammatory pathways to eliminate the pathogen, EHEC uses strategies to interfere with these signals to survive in the inflamed intestine.
Various levels of disease, from no apparent change to death, have been reported in E. In the current study, only modest histopathological changes such as increased inflammatory cells and elongation of the glands were identified Fig. S1 , Table 1 , and no mice died.
Changes in inflammatory-associated genes that are also linked to metabolic processes, such as adiponectin, leptin and thrombopoietin, indicate that metabolic processes were modulated after chronic colonization with F2, which may account for the gain in body weight. In addition to differences in weight gain, oral inoculation with O EHEC has been lethal in some mouse models.
Death was likely due to translocation of Shiga toxin into the systemic circulation after intestinal tissue damage in these lethal models. Therefore, mature ASF mice can be used as a stable non-lethal model in future studies to assess the efficacy of vaccines for reducing fecal shedding of EHEC and further explore microbe-microbe interactions.
ASF mice have not been exposed to E. To our knowledge, this is the first study to characterize a large set of inflammatory-associated genes in response to EHEC colonization in a mouse model. EHEC colonization produced a distinct gene expression pattern Table 2 , which was expected considering the large differences between non-pathogenic E. Another downregulated gene, Gzmb , is involved in apoptosis of infected cells, which may be an important aspect in the ability of EHEC to evade host immunity.
Increased expression of cytokines Bmp2 , Ccl20 , Il18 , Il23a in F2- compared to MGcolonized mice was consistent with increased inflammation observed histologically and by intestinal imaging after ProSense injection. Future studies may use RNA-seq to characterize the host transcriptome, which includes all genes rather than those already known to be involved in immunity.
Furthermore, understanding global gene expression patterns in the intestine that are affected by E. Protein-protein interactions are at the core of cellular responses, including response to infection. In the current study, chemokine interactions were the largest networks in all comparison groups, likely due to their myriad of functions during infection, e. In F2- compared to MGcolonized mice, the clustering showed interesting predictions for mechanisms of downregulation during EHEC infection. Previous research has shown that Ccl1 and Ccr8 are co-expressed in macrophages Szebeni et al.
Furthermore, Ppbp is predicted to activate expression of Ccr8 via binding based on pathway neighbor associations Kanehisa et al.
Future research should further characterize this interaction. This system can be explored in future studies to dissect the interplay between E. Gene expression analysis showed that EHEC strain F2 had a modest effect on the inflammatory response in the colon of ASF mice by differentially modulating 17 genes compared to MGcolonized mice.
Overall, these results indicate that the low-complexity ASF microbiota allowed for prolonged intestinal colonization by pathogenic and non-pathogenic strains of E.
Animal experiments were carried out in accordance with the recommendations by the Guide for the Care and Use of Laboratory Animals. Mice were acclimated for at least 2 days before each experiment. The non-pathogenic E. Bacterial suspensions were diluted twofold in PBS and 0.
Dilutions of the bacterial cultures were plated on MacConkey agar to confirm the actual inoculum. All inoculation and handling procedures were done with sterile instruments in a biosafety cabinet.
Mice were provided an irradiated diet Teklad , autoclaved water, and a h light-dark cycle was used. Animals were monitored daily for diarrhea and changes in behavior, and body weights were measured weekly. At the end of the study, mice were euthanized by carbon dioxide inhalation. Fecal samples were collected weekly over 4 weeks to determine the level of E. After 4 weeks, mice were euthanized, and contents from the duodenum, jejunum, ileum, cecum and colon were collected.
To quantify E. Stained tissue sections were examined by a board-certified veterinary pathologist blinded to the identity of the sample. Tissues were evaluated for mucosal height and scored for epithelial injury and inflammation using a rising 5-step scale with the following criteria: 0, parameter is absent; 1, parameter is present at a low level; 2, parameter is mildly increased; 3, parameter is moderately increased; 4, parameter is severe; 5, parameter is so severe that normal architecture of the tissue is distorted or lost.
Binary classification was used to describe the presence or absence of lesions observed in a given intestinal segment. Mucosal height was measured as a ratio of the length to width of the glands and only in regions of the tissue where orientation allowed for full longitudinal sections of the colonic glands to be evaluated.
ProSense is a near-infrared fluorescent agent that is optically silent but, in the presence of cathepsins primarily cathepsins B, K, L and S , is cleaved at lysine-lysine sites, which in turn releases fluorophores from quenching PerkinElmer, Waltham, MA.
ProSense has been validated to detect acute and chronic colonic inflammation in mice Ding et al. Images were analyzed in ImageJ with white set at a value of 0 and black a value of 65, An outline was drawn around each section of the intestine stomach, small intestine, cecum or large intestine and TCCF was measured as described previously McCloy et al.
Collection of colon tissues and processing was performed as previously described Henderson et al. Data were normalized and DEGs were determined for the contrasts between i uninfected versus non-pathogenic E.
All data were analyzed using GraphPad Prism version 6 software. An ANOVA followed by Tukey's test for multiple comparisons was used to compare colonization levels in fecal and intestinal samples, intestinal inflammation in ASF mice, mean body weight changes, and levels of cytokines released from colonic biopsies.
The Kruskal—Wallis test was used to compare histopathology scores and Fisher's exact test two-tailed was used to compare proportion of tissues positive for lesions. Competing interests. Author contributions. Conceptualization: M. Supplementary information. National Center for Biotechnology Information , U.
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Zhong L. Keywords: treatment, genetic mobility, pathogenesis, Escherichia , multiresistant. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.
The pathogenic E. A virotype is a particular combination of virulence genes. Important virulence factors encoded by these genes include fimbrial adhesins, enterotoxins, cytotoxins, capsule, and lipopolysaccharide, or LPS.
Pathogenic E. The following diagrams show how pathogenic E. Follow the appropriate links to learn which virulence factors are involved in each step of the disease process, and what are the important O serotypes and virotypes.
Pathogenic bacteria contaminating the environment are ingested by susceptible animals and enter the intestinal tract 1. These bacteria possess fimbrial adhesins which mediate adherence to specific receptors on the intestinal epithelial cells 2.
Resulting bacterial colonization is found mostly on the jejunal and or ileal mucosa. In some cases, the barrier that is provided by the one-cell-thick proximal tubules can be breached and bacteria can penetrate the endothelial cell to enter the bloodstream, leading to bacteraemia. This E. The incidence of infants with early-onset sepsis owing to E. As with E. These bacteria translocate from the blood to the central nervous system without apparent damage to the blood—brain barrier, which indicates a transcytosis process.
Electron micrographs imply entry by a zippering mechanism in a process that does not affect transendothelial electrical resistance This indicates that the host-cell membrane is not significantly disrupted during entry of the bacterium. Two models for studying MNEC have been developed: a monolayer of brain microvascular endothelial cells and an intact animal model using 5-day-old rats As for other E.
In genomic comparisons, the genome of E. In addition, strain RS harbours a kb plasmid, on which at least one virulence factor has been localized Some insights into the mechanism of pathogenesis of these strains have been obtained. K1 strains use S fimbriae to bind to the lumenal surfaces of brain microvascular endothelium in neonatal rats Invasion correlates with microaerobic growth and iron supplementation CNF1 is required for invasion , as is the K1 capsule, which elicits serum resistance and has antiphagocytic properties.
In an experimental model, strains that express K1 capsule proteins and those that do not were able to cross the blood—brain barrier, but only the K1-expressing strains survived As a consequence of invasion, actin cytoskeletal rearrangement occurs and tyrosine phosphorylation of focal adhesion kinase FAK and paxillin is induced In addition, a substantial list of in vivo -induced genes, including those that encode iron-acquisition systems, was compiled using in vivo expression technology IVET in conjunction with a murine model of septicaemic infection Other potential E.
Several other potential E. Among the most intriguing of these potential pathogens are strains of E. An inflammatory process, together with necrosis of the intestinal epithelium, are characteristics of necrotizing enterocolitis NEC , an important cause of mortality and long-term morbidity in pre-term infants. The ability of some E. Necrotoxic E. Strains that are known as cell-detaching E. The genes encoding CDT are infrequently present in E. However, recent information indicates that CDT can be encoded by four distinct genetic variants in E.
In at least one strain, the cdt genes are contained on a bacteriophage , which could account for the presence of this toxin in a number of different E. A poorly characterized subset of E. The initial microflora at the site of an IAI is polymicrobial, but E. A recent study indicates that a novel haem-binding protein, known as the 'haemoglobin-binding protease' Hbp , is significantly associated with E.
Purified Hbp was shown to be capable of delivering haem to B. Interestingly, Hbp is identical to Tsh, which is an autotransporter haemagglutinin that is associated with APEC, thereby indicating that this protein can contribute to at least two different infectious diseases — IAIs in humans and respiratory tract infections in poultry Mobile genetic elements.
A striking feature of pathogenic E. This was first shown more than 30 years ago with ETEC strains, in which enterotoxic activity was transferred together with a self-transmissible plasmid.
In many cases, these 'Ent' plasmids were also shown to encode antibiotic resistance. There are now numerous examples of plasmids that encode crucial virulence factors of pathogenic E. Although many of these plasmids are self-transmissible, some lack conjugation genes and can only be transferred with a conjugative plasmid. Commensal E. These additions, deletions and other genetic changes can give rise to pathogenic E. The EHEC EDL genome sequence contains 18 regions with homology to known bacteriophages, but most seem to be incomplete phage genomes Although only the Stx phage seems to be capable of lytic growth and production of infectious particles, these cryptic phage sequences enable the continued evolution of these strains by homologous recombination of phages into different chromosomal sites.
The ability to produce Stx can be readily transmitted by transduction of the genes encoding Stx phage to K or commensal E. This observation reinforces the concept that a single gene is insufficient to convert commensal E. PAIs are large genomic regions 10— kb that are present in the genomes of pathogenic strains but absent from the genomes of non-pathogenic members of the same or related species reviewed in Ref.
PAIs were first described in pathogenic E. This island is flanked by bp direct repeats, which facilitate deletion of the entire island at a relatively high frequency.
The first PAI to be described in diarrhoeagenic E. In some E. The HPI contains genes that are involved in regulation, biosynthesis and uptake of the siderophore yersiniabactin. The inverse of PAIs are 'black holes', which refers to the deletion of blocks of genes in commensal or K E.
In many EIEC strains, the cadC gene that encodes a regulator of cadA is preferentially mutated, which results in the same phenotype Although the genes encoding E. Genomic sequences. Prior to the determination of the complete genomic sequence for a pathogenic strain of E.
However, when the first pathotype was sequenced — namely two different strains of EHEC OH7 — the extent of lateral gene transfer was found to be far greater than had been anticipated. Almost as surprising was the fact that although the two strains shared a 4.
The analyses indicated that extraintestinal pathogenic E. As noted above, several studies using DNA hybridization, multilocus enzyme electrophoresis and sequencing of a small number of genes indicates that Shigella species clearly fall taxonomically within the E. The genome sequence of S.
The 4. The S. These pseudogenes arose by several mechanisms, including single-nucleotide insertions or deletions, point mutations and IS-element insertions. Interestingly, phenotypic tests that have traditionally been used to distinguish E. Whether these pseudogenes are advantageous, disadvantageous, or neutral cannot be stated at this time.
Consistent with the fact that E. In some instances, a plasmid-encoded regulator can activate transcription of chromosomal genes — for example, regulators such as the regulatory cascade formed by the EPEC plasmid-encoded regulator Per that regulates the LEE-encoded regulator, Ler Fig. Ler also regulates expression of the EspC enterotoxin that is produced by many EPEC strains and potentially other virulence factors.
Quorum sensing also regulates other factors that are potentially involved in virulence, such as flagella, through the QseBC two-component regulator In acidic conditions, the per genes are repressed by GadX, which activates the gadAB genes involved in acid resistance This dual action of GadX could prevent premature expression of virulence factors in the stomach while enhancing survival of the organism until it reaches more alkaline conditions in the small intestine where expression of virulence factors is induced.
Expression of E. A common theme among the various E. This linkage leads to induction of transcription of both toxin genes and lysis genes by certain antibiotics, causing increased toxin production, increased release of toxin by lysis and increased death in a mouse model QS is a method of intercellular communication that allows unicellular organisms such as E.
A small autoinducer AI molecule is produced by many organisms, including E. In response to this signal, expression of key virulence factors, including the LEE and Stx, is induced, thereby initiating the disease process. This regulatory mechanism can also be activated by mammalian hormones, such as adrenaline and noradrenaline, in an example of regulatory 'cross-talk' between eukaryotic and prokaryotic organisms Regulation of virulence factor expression by physical DNA rearrangements is uncommon in pathogenic E.
Transcription of the fim operon that encodes type 1 fimbriae is primarily under the control of an invertible element that contains the promoter responsible for transcription of the main structural subunit.
Individual bacterial cells either express the fimbriae over their entire surface or do not express any fimbriae. This phase variation of type 1 fimbriae is controlled at the transcriptional level by the invertible element, which is regulated by the FimB and FimE recombinases The inversion seems to be regulated during the course of infection, and the orientation of the element correlates with whether UPEC strains remain localized to the bladder.
In cystitis infections most of the strains have the invertible element in the 'on' position and express type 1 fimbriae, whereas when they leave the bladder and ascend to the kidneys to cause pyelonephritis, most of the strains have the element in the 'off' position and do not express type 1 fimbriae The regulation of type 1 fimbriae in UPEC is further complicated by cross-talk between two different adhesion operons, whereby PapB, a key regulator of the pap operon, inhibits type 1 phase variation The evolution of pathogenic E.
Using E. This genomic plasticity complicates efforts to categorize the various clusters of pathogenic E. The evolutionary process, clearly ongoing, has resulted in a highly versatile species that is capable of colonizing, multiplying in and damaging diverse environments.
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