[show abstract][hide abstract] ABSTRACT: Enterohemorrhagic Escherichia coli (EHEC) Sakai strain encodes two homologous type III effectors, EspO1-1 and EspO1-2. These EspO1s have amino acid sequence homology with Shigella OspE, which targets integrin-linked kinase to stabilize formation of focal adhesions (FAs). Like OspE, EspO1-1 was localized to FAs in EHEC-infected cells, but EspO1-2 was localized in the cytoplasm. An EHEC ΔespO1-1ΔespO1-2 double mutant induced cell rounding and FA loss in most of infected cells, but neither the ΔespO1-1 nor ΔespO1-2 single mutant did. These results suggested that EspO1-2 functioned in the cytoplasm by a different mechanism from EspO1-1 and OspE. Since several type III effectors modulate Rho GTPase, which contributes to FA formation, we investigated whether EspO1-2 modulates the function of these type III effectors. We identified a direct interaction between EspO1-2 and EspM2, which acts as a RhoA guanine nucleotide exchange factor. Upon ectopic co-expression, EspO1-2 co-localized with EspM2 in the cytoplasm and suppressed EspM2-mediated stress fiber formation. Consistent with these findings, an ΔespO1-1ΔespO1-2ΔespM2 triple mutant did not induce cell rounding in epithelial cells. These results indicated that EspO1-2 interacted with EspM2 to regulate EspM2-mediated RhoA activity and stabilize FA formation during EHEC infection.
PLoS ONE 01/2013; 8(2):e55960. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Shiga toxin-producing Escherichia coli (STEC) is one of the most important groups of food-borne pathogens, and STEC strains belonging to the serotype O103:H2 can cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. STEC O103:non-H2 strains are also sometimes isolated from human patients, but their genetic characteristics and role in significant human enteric disease are not yet understood. Here, we investigated 17 STEC O103:non-H2 strains, including O103:H11, O103:H25, O103:HUT (UT [untypeable]), and O103:H- (nonmotile) isolated in Japan, and their characteristics were compared to those of STEC O103:H2 and other serotype STEC strains. Sequence analyses of fliC and eae genes revealed that strains possessed any of the following combinations: fliC-H2/eae-epsilon, fliC-H11/eae-beta1, and fliC-H25/eae-theta, where fliC-H2, -H11, and -H25 indicate fliC genes encoding H2, H11, and H25 flagella antigens, respectively, and eae-epsilon, -beta1, and -theta indicate eae genes encoding epsilon, beta1, and theta subclass intimins, respectively. Phylogenetic analysis based on the sequences of seven housekeeping genes demonstrated that the O103:H11/[fliC-H11] and O103:H25/[fliC-H25] strains formed two distinct groups, different from that of the O103:H2/[fliC-H2] strains. Interestingly, a group consisting of O103:H11 strains was closely related to STEC O26:H11, which is recognized as a most important non-O157 serotype, suggesting that the STEC O103:H11 and STEC O26:H11 clones evolved from a common ancestor. The multiplex PCR system for the rapid typing of STEC O103 strains described in the present study may aid clinical and epidemiological studies of the STEC O103:H2, O103:H11, and O103:H25 groups. In addition, our data provide further insights into the high variability of STEC stains with emerging new serotypes.
Journal of clinical microbiology 06/2012; 50(9):2894-900. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Studies of enterohemorrhagic Escherichia coli (EHEC) infection mechanisms using mammals require large numbers of animals and are both costly and associated with ethical problems. Here, we evaluated the pathogenic mechanisms of EHEC in the silkworm model. Injection of a clinically isolated EHEC O157:H7 Sakai into either the silkworm hemolymph or intraperitoneal fluid of mice killed the host animals. EHEC O157:H7 Sakai deletion mutants of the rfbE gene, which encodes perosamine synthetase, a monosaccharide component synthetase of the O-antigen, or deletion mutants of the waaL gene, which encodes O-antigen ligase against the lipid A-core region of lipopolysaccharide (LPS), had attenuated killing ability in both silkworms and mice. Introduction of the rfbE gene or the waaL gene into the respective mutants restored the killing ability in silkworms. Growth of both mutants was inhibited by a major antimicrobial peptide in the silkworm hemolymph, moricin. The viability of both mutants was decreased in swine serum. The bactericidal effect of swine serum against both mutants was inactivated by heat treatment. These findings suggest that the LPS O-antigen of EHEC O157:H7 plays an important defensive role against antimicrobial factors in the host body fluid and is thus essential to the lethal effects of EHEC in animals.
[show abstract][hide abstract] ABSTRACT: To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx(1) stx(2) genotype showed greater resistance to heat than strains of the stx(1) stx(2c) genotype; moreover, strains of the stx(1) stx(2) genotype showed greater resistance to starvation than strains of the stx(2) or stx(2c) genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx(1) stx(2) genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx(1) stx(2c) genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx(1) stx(2) genotype, have greater multiple stress resistance than do strains of other genotypes.
Applied and environmental microbiology 02/2012; 78(9):3361-8. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: This is the first report of the isolation of Shiga toxin-producing Escherichia coli (STEC) strains whose O antigens were genetically and serologically identical to those of Shigella boydii type 10, from human feces. The novel STEC O serogroup may be widespread in Japan and associated with diarrhea and hemorrhagic colitis.
Journal of clinical microbiology 08/2011; 49(10):3678-80. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: The locus of enterocyte effacement (LEE) pathogenicity island is required for the intimate adhesion of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelial cells. GrlR and GrlA are LEE-encoded negative and positive regulators, respectively. The interaction of these two regulators is important for controlling the transcription of LEE genes through Ler, a LEE-encoded central activator for the LEE. The GrlR-GrlA regulatory system controls not only LEE but also the expression of the flagellar and enterohemolysin (Ehx) genes in EHEC. Since Ehx levels were markedly induced in a grlR mutant but not in a grlR grlA double mutant and significantly increased by overexpression of GrlA in a ler mutant, GrlA is responsible for this regulation (T. Saitoh et al., J. Bacteriol. 190:4822-4830, 2008). In this study, additional investigations of the regulation of ehx gene expression determined that Ler also acts as an activator for Ehx expression without requiring GrlA function. We recently reported that the LysR-type regulator LrhA positively controls LEE expression (N. Honda et al., Mol. Microbiol. 74:1393-1411, 2009). The hemolytic activity of the lrhA mutant strain of EHEC was lower than that of the wild-type strain, and LrhA markedly induced ehx transcription in an E. coli K-12 strain, suggesting that LrhA also activates the transcription of ehx without GrlA and Ler. Gel mobility shift assays demonstrated that Ler and LrhA directly bind to the regulatory region of ehxC. Together, these results indicate that transcription of ehx is positively regulated by Ler, GrlA, and LrhA, which all act as positive regulators for LEE expression.
Infection and immunity 08/2011; 79(11):4628-37. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genotypes of Shiga toxin-producing Escherichia coli (STEC) O157 isolated from humans and cattle were analyzed by uni- and multivariable logistic regression, and population structure methods, to gain insight into transmission and the nature of human infection. Eleven genotyping assays, including PCR typing of five virulence factors (stx(1), stx(2), stx(2c), eae, and ehxA) and a lineage-specific polymorphism assay using six markers (LSPA6), were considered in the analyses. The prevalence of the stx(1), stx(2), and stx(2c) virulence factors was significantly different between human and cattle isolates. However, multivariable regression revealed that the presence of only the stx(2) gene was significantly associated with human isolates after controlling for confounding effects. LSPA6 typing demonstrated an apparent difference in the distribution of LSPA6 lineages between human and cattle isolates and a strong association between stx genotypes and LSPA6 genotypes. Population genetics tools identified three genetically distinct clusters of STEC O157. Each cluster was characterized by stx genotypes and LSPA6 genotypes. The human isolates typically comprised LSPA6 lineage I with stx(1) stx(2) strains and LSPA6 lineage I/II with stx(2c) or stx(2) stx(2c) strains [corrected]. In contrast, the cattle isolates comprised LSPA6 lineage II strains withstx(2c) or stx(1) stx(2c) strains [corrected] in addition to the clusters identified for the human isolates. Our analyses provide new evidence that the stx(2) gene is the most distinctive feature in human isolates compared to cattle isolates in Japan, and only a subset of the genetically diverse population isolated from cattle is involved in human illnesses. Our results may contribute to international comparisons and risk assessments of STEC O157.
Journal of clinical microbiology 02/2011; 49(4):1495-500. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Enterohemorrhagic Escherichia coli (EHEC), a food- and waterborne pathogen, causes diarrhea, hemorrhagic colitis, and life-threatening HUS. MLVA is a newly developed and widely accepted genotyping tool. An MLVA system for EHEC O157 involving nine genomic loci has already been established. However, the present study revealed that the above-mentioned MLVA system cannot analyze EHEC O26 and O111 isolates-the second and third most dominant EHEC serogroups in Japan, respectively. Therefore, with several modifications to the O157 system and the use of nine additional loci, we developed an expanded MLVA system applicable to EHEC O26, O111, and O157. Our MLVA system had a relatively high resolution power for each of the three serogroups: Simpson's index of diversity was 0.991 (95% CI = 0.989-0.993), 0.988 (95% CI, 0.986-0.990), and 0.986 (95% CI, 0.979-0.993) for O26, O111, and O157, respectively. This system also detected outbreak-related isolates; the isolates collected during each of the 12 O26 and O111 outbreaks formed unique clusters, and most of the repeat copy numbers among the isolates collected during the same outbreak exhibited no or single-locus variations. These results were comparable to those of cluster analyses based on PFGE profiles. Therefore, our system can complement PFGE analysis-the current golden method. Because EHEC strains of three major serogroups can be rapidly analyzed on a single platform with our expanded MLVA system, this system could be widely used in molecular epidemiological studies of EHEC infections.
Microbiology and Immunology 10/2010; 54(10):569-77. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Serratia marcescens is a gram-negative bacterium and often causes nosocomial infections. There have been few studies of the virulence factors of this bacterium. The only S. marcescens hemolytic and cytotoxic factor reported, thus far, is the hemolysin ShlA.
An S. marcescens shlAB deletion mutant was constructed and shown to have no contact hemolytic activity. However, the deletion mutant retained hemolytic activity on human blood agar plates, indicating the presence of another S. marcescens hemolytic factor. Functional cloning of S. marcescens identified a phospholipase A (PhlA) with hemolytic activity on human blood agar plates. A phlAB deletion mutant lost hemolytic activity on human blood agar plates. Purified recombinant PhlA hydrolyzed several types of phospholipids and exhibited phospholipase A1 (PLA1), but not phospholipase A2 (PLA2), activity. The cytotoxic and hemolytic activities of PhlA both required phospholipids as substrates.
We have shown that the S. marcescens phlA gene produces hemolysis on human blood agar plates. PhlA induces destabilization of target cell membranes in the presence of phospholipids. Our results indicated that the lysophospholipids produced by PhlA affected cell membranes resulting in hemolysis and cell death.
[show abstract][hide abstract] ABSTRACT: Escherichia coli SE15 (O150:H5) is a human commensal bacterium recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2, which includes the majority of extraintestinal pathogenic E. coli. Here, we report the finished and annotated genome sequence of this organism.
Journal of bacteriology 12/2009; 192(4):1165-6. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Summary Genes essential for eliciting pathogenicity of enterohemorrhagic Escherichia coli are located within the locus of enterocyte effacement (LEE). Expression of LEE genes is positively regulated by paralogues PchA, PchB and PchC, which are encoded by separate loci of the chromosome. To elucidate the underlying regulatory mechanism, we screened transposon mutants exhibiting reduced expression of pchA, transcription level of which is highest among the pch genes. Here, we report that the LysR-homologue A (LrhA) positively regulated the transcription of pchA and pchB. A deletion in lrhA reduced the transcription levels of pchA and pchB to different degrees, and also reduced the expression of LEE-coded type 3-secreted protein, EspB. Expression of LrhA from a plasmid restored and markedly increased the transcription levels of pchA and pchB respectively, and highly induced EspB expression. Deletion analysis of the regulatory region showed that both promoter-proximal (-195 to +88) and promoter-distal (-418 to -392 for pchA and -391 to -375 for pchB) sequences were required for the LrhA-mediated upregulation of pchA and pchB genes. Purified His(6)-LrhA protein differentially bound to the regulatory regions of pchA/B, suggesting that direct regulation of pchA and pchB genes by LrhA in turn controls the expression of LEE genes.
[show abstract][hide abstract] ABSTRACT: Enterohemorrhagic Escherichia coli O157 (EHEC O157) is a food-borne pathogen that has raised worldwide public health concern. The development of simple and rapid strain-typing methods is crucial for the rapid detection and surveillance of EHEC O157 outbreaks. In the present study, we developed a multiplex PCR-based strain-typing method for EHEC O157, which is based on the variability in genomic location of IS629 among EHEC O157 strains. This method is very simple, in that the procedures are completed within 2 h, the analysis can be performed without the need for special equipment or techniques (requiring only conventional PCR and agarose gel electrophoresis systems), the results can easily be transformed into digital data, and the genes for the major virulence markers of EHEC O157 (the stx(1), stx(2), and eae genes) can be detected simultaneously. Using this method, 201 EHEC O157 strains showing different XbaI digestion patterns in pulsed-field gel electrophoresis (PFGE) analysis were classified into 127 types, and outbreak-related strains showed identical or highly similar banding patterns. Although this method is less discriminatory than PFGE, it may be useful as a primary screening tool for EHEC O157 outbreaks.
Journal of clinical microbiology 08/2009; 47(9):2888-94. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: The prevalence of STEC in Japan was examined using rectal stool samples taken from 932 healthy dairy cows from 123 farms in 11 prefectures between 2006 and 2007. Screening with stx-PCRs revealed the prevalence to be 30.4% (283 animals), and STEC strains were isolated from 111 animals. Although ten O-serogroups (O8, O22, O84, O103, O111, O113, O116, O136, O153 and O157) were the major O-serogroup in healthy dairy cows in Japan in 1998, half of the 118 selected STEC strains were serotyped as O2, O8, O26, O153, or O163 in this study. Twenty-eight of the 118 STEC strains (24%) showed resistance to some conventional drugs, such as dihydrostreptomycin, oxytetracycline and aminobenzylpenicillin. Although STEC prevalence in cows decreased from 17% to 12%, the antimicrobial resistance ratio increased from 8.7% to 24% in the past decade in Japan.
Journal of Veterinary Medical Science 04/2009; 71(3):363-6. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: We sequenced and analyzed the genome of a commensal Escherichia coli (E. coli) strain SE11 (O152:H28) recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B1. SE11 harbored a 4.8 Mb chromosome encoding 4679 protein-coding genes and six plasmids encoding 323 protein-coding genes. None of the SE11 genes had sequence similarity to known genes encoding phage- and plasmid-borne virulence factors found in pathogenic E. coli strains. The comparative genome analysis with the laboratory strain K-12 MG1655 identified 62 poorly conserved genes between these two non-pathogenic strains and 1186 genes absent in MG1655. These genes in SE11 were mostly encoded in large insertion regions on the chromosome or in the plasmids, and were notably abundant in genes of fimbriae and autotransporters, which are cell surface appendages that largely contribute to the adherence ability of bacteria to host cells and bacterial conjugation. These data suggest that SE11 may have evolved to acquire and accumulate the functions advantageous for stable colonization of intestinal cells, and that the adhesion-associated functions are important for the commensality of E. coli in human gut habitat.
DNA Research 11/2008; 15(6):375-86. · 4.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: The pathogenicity island termed locus of enterocyte effacement (LEE) encodes a type 3 protein secretion system, whose function is required for full virulence of enterohemorrhagic Escherichia coli (EHEC). GrlR and GrlA are LEE-encoded negative and positive regulators, respectively, for controlling transcription of the ler gene, which encodes a central activator of LEE gene expression. We previously reported that the GrlR-GrlA regulatory system controls not only the LEE genes but also flagellar gene expression in EHEC (S. Iyoda et al., J. Bacteriol. 188:5682-5692, 2006). In order to further explore virulence-related genes under the control of the GrlR-GrlA regulatory system, we characterized a grlR-deleted EHEC O157 strain, which was found to have high and low levels of expression of LEE and flagellar genes, respectively. We report here that the grlR deletion significantly induced enterohemolysin (Ehx) activity of EHEC O157 on plates containing defibrinated sheep erythrocytes. Ehx levels were not induced in the grlR grlA double mutant strain but increased markedly by overexpression of GrlA even in the ler mutant, indicating that GrlA is responsible for this regulation. Ehx of the EHEC O157 Sakai strain is encoded by the ehxCABD genes, which are carried on the large plasmid pO157. The expression of ehxC fused with FLAG tag or a promoterless lacZ gene on pO157 was significantly induced under conditions in which GrlA was overproduced. These results together suggest that GrlA acts as a positive regulator for the ehx transcription in EHEC.
Journal of bacteriology 08/2008; 190(14):4822-30. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.
Japanese journal of infectious diseases 02/2008; 61(1):58-64. · 1.51 Impact Factor