Polyphosphate Kinase 1 Is a Pathogenesis Determinant in Enterohemorrhagic Escherichia coli O157:H7

The ppk1 gene encodes polyphosphate kinase (PPK1), which is the major catalytic enzyme that Escherichia coli utilizes to synthesize inorganic polyphosphate (polyP). The aim of this study was to explore the role of PPK1 in the pathogenesis of Enterohemorrhagic E. coli O157:H7 (EHEC O157:H7). An isogenic in-frame ppk1 deletion mutant (Δppk1) and ppk1 complemented mutant (Cppk1) were constructed and characterized in comparison to wild-type (WT) EHEC O157:H7 strain EDL933w by microscope observation and growth curve analysis. Survival rates under heat stress and acid tolerance, both of which the bacteria would face during pathogenesis, were compared among the three strains. LoVo cells and a murine model of intestinal colitis were used as the in vitro and in vivo models, respectively, to evaluate the effect of PPK1 on adhesion and invasion during the process of pathogenesis. Real-time reverse-transcription PCR of regulatory gene rpoS, adhesion gene eae, and toxin genes stx1 and stx2 was carried out to corroborate the results from the in vitro and in vivo models. The ppk1 deletion mutant exhibited disrupted polyP levels, but not morphology and growth characteristics. The survival rate of the Δppk1 strain under stringent environmental conditions was lower as compared with WT and Cppk1. The in vitro assays showed that deletion of the ppk1 gene reduced the adhesion, formation of attaching and effacing (A/E) lesions, and invasive ability of EHEC O157:H7. Moreover, the virulence of the Δppk1 in BALB/c mice was weaker as compared with the other two strains. Additionally, mRNA expression of rpoS, eae, stx1 and stx2 were consistent with the in vitro and in vivo results. In conclusion: EHEC O157:H7 requires PPK1 for both survival under harsh environmental conditions and virulence in vivo.

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Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

Applied and Environmental Microbiology ◽

10.1128/aem.02937-06 ◽

2007 ◽

Vol 73 (10) ◽

pp. 3144-3150 ◽

Cited By ~ 140

Author(s):

Martina Bielaszewska ◽

Rita Prager ◽

Robin Köck ◽

Alexander Mellmann ◽

Wenlan Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Gene Loss ◽

Pulsed Field Gel Electrophoresis ◽

Enterohemorrhagic Escherichia Coli ◽

Toxin Gene ◽

Shiga Toxins ◽

E Coli

ABSTRACT Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx 2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.

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Enterohemorrhagic Escherichia coli Colonization of Human Colonic EpitheliumIn VitroandEx Vivo

Infection and Immunity ◽

10.1128/iai.02928-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 942-949 ◽

Cited By ~ 35

Author(s):

Steven B. Lewis ◽

Vivienne Cook ◽

Richard Tighe ◽

Stephanie Schüller

Keyword(s):

Escherichia Coli ◽

Human Colon ◽

Enterohemorrhagic Escherichia Coli ◽

Care Needs ◽

T84 Cells ◽

Host Cell Membrane ◽

Content Type ◽

Colonic Biopsy

EnterohemorrhagicEscherichia coli(EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by usingin vitroorgan culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect thein vivosituation.

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ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in Enterohemorrhagic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.12.4086-4094.2005 ◽

2005 ◽

Vol 187 (12) ◽

pp. 4086-4094 ◽

Cited By ~ 71

Author(s):

Sunao Iyoda ◽

Haruo Watanabe

Keyword(s):

Escherichia Coli ◽

Protein Secretion ◽

Deletion Mutant ◽

Regulatory Gene ◽

Secretion System ◽

Enterohemorrhagic Escherichia Coli ◽

Multicopy Plasmid ◽

Type Iii ◽

Protein Secretion System ◽

Type Iii Protein Secretion

ABSTRACT Expression of the type III protein secretion system (TTSS), encoded in the locus of enterocyte effacement (LEE) of enterohemorrhagic Escherichia coli (EHEC), has been shown to be controlled by various regulators. In a search for additional regulatory genes, we identified a DNA fragment containing clpX and clpP that has a positive regulatory effect on LEE expression in EHEC O157. The expression of LEE-encoded Esp proteins was significantly reduced in a clpXP deletion mutant. Deletion of grlR, a negative regulatory gene within LEE, markedly increased LEE expression even in the clpXP mutant. To verify the regulatory mechanism of GrlR expression, a chromosomal epitope-tagged allele of grlR (grlR-FLAG) was constructed. GrlR-FLAG expression was increased significantly in the clpXP deletion mutant, suggesting that the GrlR level is under the control of ClpXP, and this regulation is critical for the ClpXP-dependent expression of LEE in EHEC. Deletion of rpoS, the gene encoding a stationary-phase-inducing sigma factor that is a substrate for ClpXP protease, partially restored LEE expression in the clpXP mutant. A multicopy plasmid carrying rpoS strongly repressed expression of Esp proteins, suggesting that positive regulation by ClpXP is partially mediated through a negative effect of RpoS on LEE expression. We also found that rpoS deletion induces transcription of pchA, which encodes one of the positive regulators for LEE expression in EHEC. These results suggest that ClpXP controls expression of LEE through the regulation of RpoS and GrlR levels in EHEC.

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Contributions of O Island 48 to Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Epithelial Cells In Vitro and in Ligated Pig Ileal Loops

Applied and Environmental Microbiology ◽

10.1128/aem.00507-09 ◽

2009 ◽

Vol 75 (18) ◽

pp. 5779-5786 ◽

Cited By ~ 35

Author(s):

Xianhua Yin ◽

Roger Wheatcroft ◽

James R. Chambers ◽

Bianfang Liu ◽

Jing Zhu ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cluster Formation ◽

Gene Clusters ◽

Enterohemorrhagic Escherichia Coli ◽

Wild Type ◽

The Difference ◽

Large Clusters

ABSTRACT O island 48 (OI-48) of Escherichia coli consists of three functional gene clusters that encode urease, tellurite resistance (Ter), and putative adhesins Iha and AIDA-1. The functions of these clusters in enterohemorrhagic E. coli (EHEC) O157:H7 infection are unknown. Deletion mutants for these three regions were constructed and evaluated for their ability to adhere to epithelial cells in vitro and in ligated pig ileal loops. Deletion of the Ter gene cluster reduced the ability of the organism to adhere to and form large clusters on IPEC-J2 and HEp-2 cells. Complementation of the mutation by introducing the wild-type ter genes restored adherence and large-cluster formation. Tests in ligated pig ileal loops showed a decrease in colonization by the Ter-negative mutant, but the difference was not significant compared to colonization by the wild type (26.4% ± 21.2% versus 40.1% ± 19.1%; P = 0.168). The OI-48 aidA gene deletion had no effect on adherence in vitro or in vivo. Deletion of the iha and ureC genes had no effect on adherence in vitro but significantly reduced the colonization of EHEC O157:H7 in the ligated pig intestine. These data suggest that Ter, Iha, and urease may contribute to EHEC O157:H7 pathogenesis by promoting adherence of the pathogen to the host intestinal epithelium.

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Escherichia coli mhpR gene expression is regulated by catabolite repression mediated by the cAMP–CRP complex

Microbiology ◽

10.1099/mic.0.043620-0 ◽

2011 ◽

Vol 157 (2) ◽

pp. 593-600 ◽

Cited By ~ 2

Author(s):

I. Manso ◽

J. L. García ◽

B. Galán

Keyword(s):

Escherichia Coli ◽

Regulatory Gene ◽

Regulatory System ◽

Promoter Sequence ◽

Catabolic Repression ◽

Dnase I Footprinting ◽

Absolute Requirement

The expression of the mhp genes involved in the degradation of the aromatic compound 3-(3-hydroxyphenyl)propionic acid (3HPP) in Escherichia coli is dependent on the MhpR transcriptional activator at the Pa promoter. This catabolic promoter is also subject to catabolic repression in the presence of glucose mediated by the cAMP–CRP complex. The Pr promoter drives the MhpR-independent expression of the regulatory gene. In vivo and in vitro experiments have shown that transcription from the Pr promoter is downregulated by the addition of glucose and this catabolic repression is also mediated by the cAMP–CRP complex. The activation role of the cAMP–CRP regulatory system was further investigated by DNase I footprinting assays, which showed that the cAMP–CRP complex binds to the Pr promoter sequence, protecting a region centred at position −40.5, which allowed the classification of Pr as a class II CRP-dependent promoter. Open complex formation at the Pr promoter is observed only when RNA polymerase and cAMP–CRP are present. Finally, by in vitro transcription assays we have demonstrated the absolute requirement of the cAMP–CRP complex for the activation of the Pr promoter.

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Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli

Nature Communications ◽

10.1038/s41467-020-20355-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Cheng-Rung Huang ◽

Cheng-Ju Kuo ◽

Chih-Wen Huang ◽

Yu-Ting Chen ◽

Bang-Yu Liu ◽

...

Keyword(s):

Escherichia Coli ◽

Enterohemorrhagic Escherichia Coli ◽

Intestinal Cell ◽

Intestinal Cells ◽

Cyclin Dependent Kinase ◽

C Elegans ◽

Mitotic Cyclin ◽

Human Intestinal Cells

AbstractEnterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

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A Novel Small RNA Promotes Motility and Virulence of Enterohemorrhagic Escherichia coli O157:H7 in Response to Ammonium

mBio ◽

10.1128/mbio.03605-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Tianyuan Jia ◽

Bin Liu ◽

Huiqian Mu ◽

Chengqian Qian ◽

Lu Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Environmental Changes ◽

Bacterial Colonization ◽

Rabbit Model ◽

Enterohemorrhagic Escherichia Coli ◽

Host Cells ◽

Bacterial Motility ◽

Future Research

ABSTRACT Enterohemorrhagic Escherichia coli serotype O157:H7 (O157) is a critical, foodborne, human intestinal pathogen that causes severe acute hemorrhagic diarrhea, abdominal cramping, and even death. Small RNAs (sRNAs) are noncoding regulatory molecules that sense environmental changes and trigger various virulence-related signaling pathways; however, few such sRNAs have been identified in O157. Here, we report a novel sRNA, EsrF that senses high ammonium concentrations in the colon and enhances O157 pathogenicity by promoting bacterial motility and adhesion to host cells. Specifically, EsrF was found to directly interact with the 5′ untranslated regions of the flagellar biosynthetic gene, flhB, mRNA and increase its abundance, thereby upregulating expression of essential flagellar genes, including flhD, flhC, fliA, and fliC, leading to elevated O157 motility and virulence. Meanwhile, an infant rabbit model of O157 infection showed that deletion of esrF and flhB significantly attenuates O157 pathogenicity. Furthermore, NtrC—the response regulator of the NtrC/B two-component system—was found to exert direct, negative regulation of esrF expression. Meanwhile, high ammonium concentrations in the colon release the inhibitory effect of NtrC on esrF, thereby enhancing its expression and subsequently promoting bacterial colonization in the host colon. Our work reveals a novel, sRNA-centered, virulence-related signaling pathway in O157 that senses high ammonium concentrations. These findings provide novel insights for future research on O157 pathogenesis and targeted treatment strategies. IMPORTANCE The process by which bacteria sense environmental cues to regulate their virulence is complex. Several studies have focused on regulating the expression of the locus of enterocyte effacement pathogenicity island in the typical gut pathogenic bacterium, O157. However, few investigations have addressed the regulation of other virulence factors in response to intestinal signals. In this study, we report our discovery of a novel O157 sRNA, EsrF, and demonstrate that it contributed to bacterial motility and virulence in vitro and in vivo through the regulation of bacterial flagellar synthesis. Furthermore, we show that high ammonium concentrations in the colon induced esrF expression to promote bacterial virulence by releasing the repression of esrF by NtrC. This study highlights the importance of sRNA in regulating the motility and pathogenicity of O157.

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Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence

Frontiers in Microbiology ◽

10.3389/fmicb.2018.00947 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 4

Author(s):

Thomas Douëllou ◽

Wessam Galia ◽

Stéphane Kerangart ◽

Thierry Marchal ◽

Nadège Milhau ◽

...

Keyword(s):

Escherichia Coli ◽

Milk Fat ◽

Enterohemorrhagic Escherichia Coli ◽

Fat Globules ◽

Milk Fat Globules

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Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Clinical and Vaccine Immunology ◽

10.1128/cvi.00495-09 ◽

2010 ◽

Vol 17 (4) ◽

pp. 529-536 ◽

Cited By ~ 13

Author(s):

Robert L. G. Rojas ◽

Priscila A. D. P. Gomes ◽

Leticia V. Bentancor ◽

Maria E. Sbrogio-Almeida ◽

Sérgio O. P. Costa ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Plasmid Stability ◽

Present Report ◽

Enterohemorrhagic Escherichia Coli ◽

Bacterial Strains ◽

Gut Colonization ◽

Serovar Typhimurium

ABSTRACT Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2ΔAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2ΔAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2ΔAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

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Cortactin Recruitment by Enterohemorrhagic Escherichia coli O157:H7 during Infection In Vitro and Ex Vivo

Infection and Immunity ◽

10.1128/iai.00140-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4669-4676 ◽

Cited By ~ 7

Author(s):

Aurelie Mousnier ◽

Andrew D. Whale ◽

Stephanie Schüller ◽

John M. Leong ◽

Alan D. Phillips ◽

...

Keyword(s):

Escherichia Coli ◽

Actin Polymerization ◽

Cultured Cells ◽

Ex Vivo ◽

Enterohemorrhagic Escherichia Coli ◽

T3ss Effector ◽

Mucosal Surfaces ◽

N Terminus

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspFU. Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.

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