scholarly journals Involvement of PatE, a Prophage-Encoded AraC-Like Regulator, in the Transcriptional Activation of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933

2012 ◽  
Vol 78 (15) ◽  
pp. 5083-5092 ◽  
Author(s):  
Jennifer K. Bender ◽  
Judyta Praszkier ◽  
Matthew J. Wakefield ◽  
Kathryn Holt ◽  
Marija Tauschek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Infectious Dose ◽  
Genes Encoding

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach.In silicoanalysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene,patE, for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis inE. colishowed that the expression ofpatEis induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperonehdeAby binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.

scholarly journals Control of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933 by PsrB, a Prophage-Encoded AraC-Like Regulator

2014 ◽  
Vol 83 (1) ◽  
pp. 346-353 ◽  
Author(s):  
Ji Yang ◽  
Thomas W. Russell ◽  
Dianna M. Hocking ◽  
Jennifer K. Bender ◽  
Yogitha N. Srikhanta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Resistance ◽  
Transcriptional Regulators ◽  
Coli Strain ◽  
Enterohemorrhagic Escherichia Coli ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
E Coli ◽  
Acidic Conditions

EnterohemorrhagicEscherichia coli(EHEC) O157:H7 causes bloody diarrhea and hemolytic-uremic syndrome (HUS) and is the most prevalentE. coliserotype associated with food-borne illness worldwide. This pathogen is transmitted via the fecal-oral route and has a low infectious dose that has been estimated to be between 10 and 100 cells. We and others have previously identified three prophage-encoded AraC-like transcriptional regulators, PatE, PsrA, and PsrB in the EHEC O157:H7 EDL933 strain. Our analysis showed that PatE plays an important role in facilitating survival of EHEC under a number of acidic conditions, but the contribution of PsrA and PsrB to acid resistance (AR) was unknown. Here, we investigated the involvement of PsrA and PsrB in the survival ofE. coliO157:H7 in acid. Our results showed that PsrB, but not PsrA, enhanced the survival of strain EDL933 under various acidic conditions. Transcriptional analysis using promoter-lacZreporters and electrophoretic mobility shift assays demonstrated that PsrB activates transcription of thehdeAoperon, which encodes a major acid stress chaperone, by interacting with its promoter region. Furthermore, using a mouse model, we showed that expression of PsrB significantly enhanced the ability of strain EDL933 to overcome the acidic barrier of the mouse stomach. Taken together, our results indicate that EDL933 acquired enhanced acid tolerance via horizontally acquired regulatory genes encoding transcriptional regulators that activate its AR machinery.

scholarly journals RegR Virulence Regulon of Rabbit-Specific Enteropathogenic Escherichia coli Strain E22

2013 ◽  
Vol 81 (4) ◽  
pp. 1078-1089 ◽  
Author(s):  
Yogitha N. Srikhanta ◽  
Dianna M. Hocking ◽  
Judyta Praszkier ◽  
Matthew J. Wakefield ◽  
Roy M. Robins-Browne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Bioinformatic Analysis ◽  
Coli Strain ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Gene Target ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

ABSTRACTAraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenicEscherichia coli(EPEC), enterotoxigenicE. coli, enteroaggregativeE. coli, andCitrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, ofC. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target,sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression ofsefAby binding to a region upstream of thesefApromoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.

scholarly journals Global Regulator of Virulence A (GrvA) Coordinates Expression of Discrete Pathogenic Mechanisms in Enterohemorrhagic Escherichia coli through Interactions with GadW-GadE

2015 ◽  
Vol 198 (3) ◽  
pp. 394-409 ◽  
Author(s):  
Jason K. Morgan ◽  
Ronan K. Carroll ◽  
Carly M. Harro ◽  
Khoury W. Vendura ◽  
Lindsey N. Shaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Sequencing Analysis ◽  
Global Regulator ◽  
Altered Expression ◽  
Content Type ◽  
Pathogenic Mechanisms ◽  
Gut Colonization ◽  
Intestinal Pathogens

ABSTRACTGlobal regulator of virulence A (GrvA) is a ToxR-family transcriptional regulator that activates locus of enterocyte effacement (LEE)-dependent adherence in enterohemorrhagicEscherichia coli(EHEC). LEE activation by GrvA requires the Rcs phosphorelay response regulator RcsB and is sensitive to physiologically relevant concentrations of bicarbonate, a known stimulant of virulence systems in intestinal pathogens. This study determines the genomic scale of GrvA-dependent regulation and uncovers details of the molecular mechanism underlying GrvA-dependent regulation of pathogenic mechanisms in EHEC. In agrvA-null background of EHEC strain TW14359, RNA sequencing analysis revealed the altered expression of over 700 genes, including the downregulation of LEE- and non-LEE-encoded effectors and the upregulation of genes for glutamate-dependent acid resistance (GDAR). Upregulation of GDAR genes corresponded with a marked increase in acid resistance. GrvA-dependent regulation of GDAR and the LEE requiredgadE, the central activator of GDAR genes and a direct repressor of the LEE. Control ofgadEby GrvA was further determined to occur through downregulation of thegadEactivator GadW. This interaction of GrvA with GadW-GadE represses the acid resistance phenotype, while it concomitantly activates the LEE-dependent adherence and secretion of immune subversion effectors. The results of this study significantly broaden the scope of GrvA-dependent regulation and its role in EHEC pathogenesis.IMPORTANCEEnterohemorrhagicEscherichia coli(EHEC) is an intestinal human pathogen causing acute hemorrhagic colitis and life-threatening hemolytic-uremic syndrome. For successful transmission and gut colonization, EHEC relies on the glutamate-dependent acid resistance (GDAR) system and a type III secretion apparatus, encoded on the LEE pathogenicity island. This study investigates the mechanism whereby the DNA-binding regulator GrvA coordinates activation of the LEE with repression of GDAR. Investigating how these systems are regulated leads to an understanding of pathogenic behavior and novel strategies aimed at disease prevention and control.

scholarly journals Global Analysis of Posttranscriptional Regulation by GlmY and GlmZ in Enterohemorrhagic Escherichia coli O157:H7

2015 ◽  
Vol 83 (4) ◽  
pp. 1286-1295 ◽  
Author(s):  
Charley C. Gruber ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Rna Sequencing ◽  
Posttranscriptional Regulation ◽  
Global Analysis ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Global Changes ◽  
Content Type ◽  
Enterocyte Effacement ◽  
K 12

EnterohemorrhagicEscherichia coli(EHEC) is a significant human pathogen and is the cause of bloody diarrhea and hemolytic-uremic syndrome. The virulence repertoire of EHEC includes the genes within the locus of enterocyte effacement (LEE) that are largely organized in five operons,LEE1toLEE5, which encode a type III secretion system, several effectors, chaperones, and regulatory proteins. In addition, EHEC also encodes several non-LEE-encoded effectors and fimbrial operons. The virulence genes of this pathogen are under a large amount of posttranscriptional regulation. The small RNAs (sRNAs) GlmY and GlmZ activate the translation of glucosamine synthase (GlmS) inE. coliK-12, and in EHEC they destabilize the 3′ fragments of theLEE4andLEE5operons and promote translation of the non-LEE-encoded effector EspFu. We investigated the global changes of EHEC gene expression governed by GlmY and GlmZ using RNA sequencing and gene arrays. This study extends the known effects of GlmY and GlmZ regulation to show that they promote expression of the curli adhesin, repress the expression of tryptophan metabolism genes, and promote the expression of acid resistance genes and the non-LEE-encoded effector NleA. In addition, seven novel EHEC-specific sRNAs were identified using RNA sequencing, and three of them—sRNA56, sRNA103, and sRNA350—were shown to regulate urease, fimbria, and the LEE, respectively. These findings expand the knowledge of posttranscriptional regulation in EHEC.

scholarly journals Evolutionary Silence of the Acid Chaperone Protein HdeB in Enterohemorrhagic Escherichia coli O157:H7

2011 ◽  
Vol 78 (4) ◽  
pp. 1004-1014 ◽  
Author(s):  
Michelle Q. Carter ◽  
Jacqueline W. Louie ◽  
Clifton K. Fagerquist ◽  
Omar Sultan ◽  
William G. Miller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Resistance ◽  
Significant Loss ◽  
Enterohemorrhagic Escherichia Coli ◽  
Survival Strategies ◽  
Start Codon ◽  
Divergent Evolution ◽  
Content Type ◽  
E Coli ◽  
K 12

ABSTRACTThe periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) inEscherichia coliandShigellaspp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagicE. coli(EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less inE. coliO157:H7 strains. Deletion ofhdeBdid not affect the acid survival of cells, and deletion ofhdeAled to less than a 5-fold decrease in survival. Sequence analysis of thehdeABoperon revealed a point mutation at the putative start codon of thehdeBgene in all 26E. coliO157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB inE. coliO157:H7; however, the plasmid-borne O157-hdeBwas able to restore partially the acid resistance in anE. coliO145ΔhdeABmutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude thatE. coliO157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms withinE. coli.

scholarly journals GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

2013 ◽  
Vol 80 (2) ◽  
pp. 714-722 ◽  
Author(s):  
Junhong Wei ◽  
Yuqing Tian ◽  
Guoqing Niu ◽  
Huarong Tan
Keyword(s):  
Biological Activities ◽  
Regulatory Protein ◽  
Specific Inhibitor ◽  
Biosynthetic Gene Cluster ◽  
Binding Activity ◽  
Major Facilitator Superfamily ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Inhibitor Of Protein Synthesis

ABSTRACTGougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities.gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed thatgouRplays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of thegouL-to-gouBoperon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions ofgouL,gouM, andgouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export inStreptomyces graminearus.

scholarly journals The Acyl-Homoserine Lactone Synthase YenI from Yersinia enterocolitica Modulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli O157:H7

2013 ◽  
Vol 81 (11) ◽  
pp. 4192-4199 ◽  
Author(s):  
Y N. Nguyen ◽  
Haiqing Sheng ◽  
Rambabu Dakarapu ◽  
John R. Falck ◽  
Carolyn J. Hovde ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Yersinia Enterocolitica ◽  
Virulence Gene ◽  
Acid Resistance ◽  
Homoserine Lactone ◽  
Enterohemorrhagic Escherichia Coli ◽  
Content Type ◽  
Fecal Shedding ◽  
Virulence Gene Expression

ABSTRACTThe human pathogen enterohemorrhagicEscherichia coli(EHEC) O157:H7 colonizes the rectoanal junction (RAJ) in cattle, its natural reservoir. Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses acyl-homoserine lactones (AHLs) produced by the microbiota in the rumen to activate thegadacid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine if the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express theYersinia enterocoliticaAHL synthase geneyenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. TheyenI+EHEC produces oxo-C6-homoserine lactone (oxo-C6-HSL) and had a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formationin vitrocompared to the wild type (WT). TheyenI+EHEC also activated expression of thegadgenes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT oryenI+EHEC. Although theyenI+EHEC colonized the RAJ with efficiency equal to that of the WT, there was a trend for the cattle to shed the WT strain longer than theyenI+EHEC.

scholarly journals The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion

2015 ◽  
Vol 84 (2) ◽  
pp. 537-549 ◽  
Author(s):  
Takeshi Shimizu ◽  
Kimitoshi Ichimura ◽  
Masatoshi Noda
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Enterohemorrhagic Escherichia Coli ◽  
Mobility Shift ◽  
Flagellin Gene ◽  
Content Type ◽  
Type Iii ◽  
Cpx Pathway

Although the adhesion of enterohemorrhagicEscherichia coli(EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces byE. colihas been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to thelrhApromoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection.

scholarly journals Comparative Genomics and Immunoinformatics Approach for the Identification of Vaccine Candidates for Enterohemorrhagic Escherichia coli O157:H7

2014 ◽  
Vol 82 (5) ◽  
pp. 2016-2026 ◽  
Author(s):  
Víctor A. García-Angulo ◽  
Anjana Kalita ◽  
Mridul Kalita ◽  
Luis Lozano ◽  
Alfredo G. Torres
Keyword(s):  
Escherichia Coli ◽  
Comparative Genomics ◽  
Enterohemorrhagic Escherichia Coli ◽  
Th2 Cytokines ◽  
Structural Protein ◽  
Gene Encoding ◽  
Content Type ◽  
Vaccine Candidates ◽  
Genes Encoding ◽  
K 12

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic-uremic syndrome worldwide. Thus far, there is no vaccine for humans against EHEC infections. In this study, a comparative genomics analysis was performed to identify EHEC-specific antigens useful as potential vaccines. The genes present in both EHEC EDL933 and Sakai strains but absent in nonpathogenicE. coliK-12 and HS strains were subjected to anin silicoanalysis to identify secreted or surface-expressed proteins. We obtained a total of 65 gene-encoding protein candidates, which were subjected to immunoinformatics analysis. Our criteria of selection aided in categorizing the candidates as high, medium, and low priority. Three members of each group were randomly selected and cloned into pVAX-1.Candidates were pooled accordingly to their priority group and tested for immunogenicity against EHEC O157:H7 using a murine model of gastrointestinal infection. The high-priority (HP) pool, containing genes encoding a Lom-like protein (pVAX-31), a putative pilin subunit (pVAX-12), and a fragment of the type III secretion structural protein EscC (pVAX-56.2), was able to induce the production of EHEC IgG and sIgA in sera and feces. HP candidate-immunized mice displayed elevated levels of Th2 cytokines and diminished cecum colonization after wild-type challenge. Individually tested HP vaccine candidates showed that pVAX-12 and pVAX-56.2 significantly induced Th2 cytokines and production of fecal EHEC sIgA, with pVAX-56.2 reducing EHEC cecum colonization. We describe here a bioinformatics approach able to identify novel vaccine candidates potentially useful for preventing EHEC O157:H7 infections.

scholarly journals Distinct Acid Resistance and Survival Fitness Displayed by Curli Variants of Enterohemorrhagic Escherichia coli O157:H7

2011 ◽  
Vol 77 (11) ◽  
pp. 3685-3695 ◽  
Author(s):  
Michelle Q. Carter ◽  
Maria T. Brandl ◽  
Jacqueline W. Louie ◽  
Jennifer L. Kyle ◽  
Diana K. Carychao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Resistance ◽  
Cell Aggregation ◽  
Oxygen Depletion ◽  
Growth Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Surface Attachment ◽  
Content Type ◽  
E Coli ◽  
Physiological Properties

ABSTRACTCurli are adhesive fimbriae ofEnterobacteriaceaeand are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter- and intrastrain variations in curli production are widespread in enterohemorrhagicEscherichia coliO157:H7. The relative proportions of curli-producing variants (C+) and curli-deficient variants (C−) in anE. coliO157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C+and C−subpopulations occurred mostly in response to starvation and was unidirectional from C−to C+; in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C+variants grew to higher concentrations in nutrient-limited conditions than C−variants, whereas C−variants were significantly more acid resistant than C+variants. This difference in acid resistance does not appear to be linked to the curli fimbriaeper se, since acsgAdeletion mutant in either a C+or a C−variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants ofE. coliO157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in anE. coliO157:H7 population may provide a survival strategy in which C+variants are selected in a nutrient-limited environment, whereas C−variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.

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