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Most Shiga toxin-producing Escherichia coli (STEC) infections that are associated with severe sequelae such as hemolytic uremic syndrome (HUS) are caused by attaching and effacing pathogens that carry the locus of enterocyte effacement (LEE). However, a proportion of STEC isolates that do not carry LEE have been associated with HUS. To clarify the emergence of LEE-negative STEC, we compared the genetic composition of the virulence plasmids pO113 and pO157 from LEE-negative and LEE-positive STEC, respectively. The complete nucleotide sequence of pO113 showed that several plasmid genes were shared by STEC O157:H7. In addition, allelic profi ling of the ehxA gene demonstrated that pO113 belongs to a different evolutionary lineage than pO157 and that the virulence plasmids of LEE-negative STEC strains were highly related. In contrast, multilocus sequence typing of 17 LEE-negative STEC isolates showed several clonal groups, suggesting that pathogenic LEE-negative STEC has emerged several times throughout its evolution. [ABSTRACT FROM AUTHOR]

Regulation of virulence gene expression plays a central role in the pathogenesis of enteric bacteria as they encounter diverse environmental conditions in the gastrointestinal tract of their hosts. In this study, we investigated environmental regulation of two putative virulence determinants adcA and kfc by RegA, an AraC/XylS-like regulator, from Citrobacter rodentium, and identified bicarbonate as the environmental signal which induced transcription of adcA and kfc through RegA. Primer extension experiments showed that adcA and kfc were divergently transcribed from σ70 promoters. In vivo and in vitro experiments demonstrated that bicarbonate facilitated and stabilized the binding of RegA to an operator located between the two promoters. The interaction of RegA with its DNA target resulted in the formation of a nucleosome-like structure, which evidently displaced the histone-like proteins, H-NS and StpA, from the adcA and kfc promoter regions, leading to transcriptional derepression. In addition, our results indicated that RegA also behaved as a Class I activator by directly stimulating transcription initiation by RNA polymerase. This is the first report to describe the molecular mechanism by which an environmental chemical stimulates transcription of virulence-associated genes of an enteric pathogen through an AraC/XlyS-like activator. [ABSTRACT FROM AUTHOR]

Enteropathogenic Escherichia coli induces characteristic attaching–effacing (A/E) lesions on the intestinal mucosa during infection. The locus of enterocyte effacement is essential for A/E lesion formation and encodes a type III secretion system that translocates multiple effector proteins into the host cell. Following translocation, EspI/NleA localizes to the Golgi. Using the yeast two-hybrid system (Y2HS) and PSD-95/Disk-large/ZO-1 (PDZ)-domain protein array overlays, we identified 15 putative host-interacting partners of EspI. All but two of the target proteins contained PDZ domains. Examination of the EspI amino acid sequence revealed a C-terminal consensus class I PDZ binding motif. Deletion of the last 7 amino acids of EspI to generate EspIΔC7 abrogated the Y2HS interaction between EspI and 5 of the 6 putative host cell target proteins tested. Deletion of the EspI PDZ binding motif also resulted in delayed trafficking of EspI to the Golgi. Using a mouse model of infection, we showed that Citrobacter rodentium expressing truncated EspIΔC7 was attenuated when in competition with C. rodentium expressing full-length EspI. Overall, these results suggested that EspI may modulate the virulence of A/E pathogens by binding host PDZ-domain proteins. [ABSTRACT FROM AUTHOR]

Background. It is postulated that the surge in incidence and severity of group A streptococcus (GAS) infections since the 1980s is due to the emergence of strains of GAS with increased virulence. We used active, population-based surveillance of invasive GAS disease, serologically confirmed pharyngitis, and carriage to determine whether particular strains were associated with invasive disease. Methods. Two hundred twenty GAS isolates were collected—78 invasive, 34 pharyngitis, and 108 carriage. Isolates were characterized using emm typing, random amplification of polymorphic DNA (RAPD) profiling, and superantigen genotyping. Results. emm1, emm12, and emm28 predominated in invasive disease and accounted for 30.8%, 12.8%, and 12.8% of all isolates, respectively. emm1, emm75, emm28, and emm4 were the most frequently isolated emm types in pharyngitis, and emm12 and emm1 predominated in carriage. emm12 was significantly associated with carriage rather than disease. There were no other significant associations between emm type and disease or carriage. There were no associations between any RAPD profile or superantigen genotype and invasive disease, pharyngitis, or carriage. One RAPD profile accounted for most cases of necrotizing fasciitis, which suggests that this strain might have particular features promoting connective-tissue infection. Conclusions. These data suggest that the emergence of GAS strains with increased virulence is not the main factor responsible for the surge in GAS-related infections. The prevalence of particular emm types, RAPD profiles, or superantigen genes in invasive disease may simply indicate widespread transmission of these strains in the population, rather than a particular ability to cause disease. [ABSTRACT FROM AUTHOR]

As part of a study to determine the effects of water filtration on the incidence of community-acquired gastroenteritis in Melbourne, Australia, we examined fecal samples from patients with gastroenteritis and asymptomatic persons for diarrheagenic strains of Escherichia coli. Atypical strains of enteropathogenic E. coli (EPEC) were the most frequently identified pathogens of all bacterial, viral, and parasitic agents in patients with gastroenteritis. Moreover, atypical EPEC were more common in patients with gastroenteritis (89 [12.8%] of 696) than in asymptomatic persons (11 [2.3%] of 489, p < 0.0001). Twenty-two random isolates of atypical EPEC that were characterized further showed marked heterogeneity in terms of serotype, genetic subtype, and carriage of virulence-associated determinants. Apart from the surface protein, intimin, no virulence determinant or phenotype was uniformly present in atypical EPEC strains. This study shows that atypical EPEC are an important cause of gastroenteritis in Melbourne. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica strains of biotype 1A lack the known virulence determinants of strains in other categories, including the Yersinia virulence plasmid (pYV), and several chromosomal markers of pathogenicity. For this reason, and also because Y. enterocolitica strains of biotype 1A are frequently isolated from the environment or asymptomatic individuals, these bacteria are often assumed to be avirulent. On the other hand, there is a considerable body of clinical, epidemiological and experimental evidence to indicate that at least some strains of Y. enterocolitica biotype 1A are able to cause gastrointestinal symptoms which resemble those caused by pYV-bearing strains. The availability of a number of experimental systems, including cell culture and animal models of infection, provides an opportunity to identify and characterise the essential virulence determinants of biotype 1A strains. [Copyright &y& Elsevier]

Summary We have characterized the LEE pathogenicity islands (PAIs) of two rabbit-specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H-) and 84/110-1 (O103:H2), and have compared them to homologous loci from the human enteropathogenic and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC strain, RDEC-1. All five PAIs contain a 34 kb core region that is highly conserved in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly larger (59 540 basepairs) than those of the human strains, which are less than 44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb core of 83/39 contain homologues of two putative virulence determinants, efa1 /lifA and senA . The LEE of 84/110-1 is approximately 85 kb and is located at pheV tRNA. Its core is almost identical to those of 83/39 and RDEC-1, apart from a larger espF gene, but its flanking regions contain trcA , a putative virulence determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int-phe. The LEE PAI of 84/110-1 is also flanked by short direct repeats (representing the 3′ -end of pheV tRNA), suggesting that it may be unstable. To investigate this possibility, we constructed a LEE::sacB derivative of 84/110-1 and showed that the PAI was capable of spontaneous deletion. We also showed that Int-phe can mediate site-specific integration of foreign DNA at the pheU tRNA locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI. [ABSTRACT FROM AUTHOR]

This review highlights the exciting new finding that small molecule inducers have been found for some members of the AraC family of transcriptional regulators (AFTRs) that control the expression of virulence genes of pathogenic bacteria. Although effector-mediated activation of AFTRs involved in the regulation of sugar and alkylbenzoate catabolism (e.g. AraC and XylS) is well understood, until recently little was known about effector-mediated regulation of virulence gene expression. This led to the belief that regulation of virulence by AFTRs does not involve the direct recognition of chemical environmental signals. More recently, however, a growing number of virulence-associated AFTRs have been found to directly sense environmental chemicals. Most interestingly, these environmental chemicals are abundant at the sites where the bacterial pathogen colonizes and damages its host. In this article we review recent developments in the field of environmental regulation mediated by virulence-associated AFTRs, with a focus on the sensing of environmental signals, the mechanism of gene target activation, and the effector-mediated modulation of regulator activities. [ABSTRACT FROM AUTHOR]

The misuse of antibiotics during past decades has led to pervasive antibiotic resistance in bacteria. Hence, there is an urgent need for the development of new and alternative approaches to combat bacterial infections. In most bacterial pathogens the expression of virulence is tightly regulated at the transcriptional level. Therefore, targeting pathogens with drugs that interfere with virulence gene expression offers an effective alternative to conventional antimicrobial chemotherapy. Many Gram-negative intestinal pathogens produce AraC-like proteins that control the expression of genes required for infection. In this study we investigated the prototypical AraC-like virulence regulator, RegA, from the mouse attaching and effacing pathogen, Citro-bacter rodentium, as a potential drug target. By screening a small molecule chemical library and chemical optimization, we identified two compounds that specifically inhibited the ability of RegA to activate its target promoters and thus reduced expression of a number of proteins required for virulence. Bio-physical, biochemical, genetic, and computational analyses indicated that the more potent of these two compounds, which we named regacin, disrupts the DNA binding capacity of RegA by interacting with amino acid residues within a conserved region of the DNA binding domain. Oral administration of regacin tomice, commencing 15min before or 12 h after oral inoculation with C. rodentium, caused highly significant attenuation of intestinal colonization by themouse pathogen comparable to that of an isogenic regA-deletion mutant. These findings demonstrate that chemical inhibition of the DNA binding domains of transcriptional regulators is a viable strategy for the development of antimicrobial agents that target bacterial pathogens. [ABSTRACT FROM AUTHOR]

We identified several promoters responsible for the expression of regA, which encodes a global virulence regulator in Citrobacter rodentium. Expression of some of the promoters was strongly autoactivated by RegA in conjunction with bicarbonate. Biochemical and mutational analyses were used to determine the consensus sequence of the RegA-binding sites. [ABSTRACT FROM AUTHOR]

Abstract: The global virulence regulatory protein RegA, an AraC-like regulator, controls the expression of more than 60 genes in the mouse enteric pathogen Citrobacter rodentium. In the presence of bicarbonate, RegA activates the transcription of a number of virulence determinants and inhibits the expression of a series of housekeeping genes. To elucidate the molecular mechanism by which bicarbonate stimulates RegA activity, we carried out biophysical and mutational analyses. Our data indicate that RegA exists as a dimer in solution regardless of bicarbonate concentration. A leucine zipper, located in the region downstream of the N-terminal domain, is responsible for dimerisation. The N-terminal arm itself is involved in modulating the response to bicarbonate, which appears to bind to a region comprising a series of β-sheets within the N-terminal domain. The presence of bicarbonate relieves the autoinhibition of RegA activity by its N-terminal arm. RegA is the first example of a bacterial virulence regulator that utilises the light switch mechanism, previously described for the Escherichia coli AraC protein, to respond to a gut-associated effector that controls its activity. [Copyright &y& Elsevier]

GrlA and GrlR proteins work as activator and antiactivator, respectively, of ler transcription. Thus, control of the transcriptional activities of the LEE1 (ler) promoter and the grlRA operon determines the rate of transcription of all of the LEE-encoded virulence factors. To date, only a single promoter has been identified for the grlRA operon. In this study, we showed that the non-LEE-encoded AraC-like regulatory protein RegA of C. rodentium directly stimulates transcription of the grlRA promoter by binding to an upstream region in the presence of bicarbonate ions. In addition, in vivo and in vitro transcription assays revealed a σ70 promoter that is specifically responsible for transcription of grlA. Expression from this promoter was strongly repressed by H-NS and its paralog StpA but was activated by Ler. DNase I footprinting demonstrated that Ler binds to a region upstream of the grlA promoter, whereas H-NS interacts specifically with a region extending from the grlA core promoter into its coding sequence. Together, these findings provide new insights into the environmental regulation and differential expressions of the grlR and grlA genes of C. rodentium. [ABSTRACT FROM AUTHOR]