The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae.

Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilm matrix protein (RbmC) by base-pairing with the 5' region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA down-regulates Vrp protein synthesis by base-pairing to the 5' region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation.

A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles.

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5' region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, sigma(E), suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract.

Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni.

BACKGROUND: Background: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment. RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form. CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.

A novel sRNA that modulates virulence and environmental fitness of Vibrio cholerae.

We recently described the discovery and initial functional characterization of a new sRNA, VrrA, in Vibrio cholerae O1 strain A1552. The VrrA homologs were found in all Vibrio strains whose genome sequences were reported at present. In this article, we summarize the multi-functional features of VrrA in V. cholerae pathogenesis and physiology, especially in relation to the regulation of outer membrane vesicle formation and its consequence in environmental adaptation of the bacterium. As the vrrA gene was not predicted by any of the previous bioinformatics-based genome-wide screenings for sRNA, we discuss the reasons and give suggestion on improving current bioinformatics tools.

The metalloprotease PrtV from Vibrio cholerae.

The Vibrio metalloprotease PrtV was purified from the culture supernatant of a Vibrio cholerae derivative that is deficient in several other secreted peptidases, including the otherwise abundant hemagglutinin/protease HapA. The PrtV is synthesized as a 102 kDa protein, but undergoes several N- and C-terminal processing steps during V. cholerae envelope translocation and prolonged incubation. Purified V. cholerae PrtV protease forms of 81 or 73 kDa were stabilized by calcium ions. Removal of calcium resulted in further rapid autoproteolysis. The two major products of autoproteolysis of the PrtV protease were approximately 37 and 18 kDa and could not be separated under non-denaturing conditions, indicating they are interacting domains. In an assay using cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell death. Using human blood plasma as a source of potential substrates of mammalian origin for the PrtV protease, we found that the extracellular matrix components fibronectin and fibrinogen were degraded by the enzyme. Additional tests with individual protein substrates revealed that plasminogen was also a possible target for the PrtV protease.

Sub-Optimal Treatment of Bacterial Biofilms.

Bacterial biofilm is an emerging clinical problem recognized in the treatment of infectious diseases within the last two decades. The appearance of microbial biofilm in clinical settings is steadily increasing due to several reasons including the increased use of quality of life-improving artificial devices. In contrast to infections caused by planktonic bacteria that respond relatively well to standard antibiotic therapy, biofilm-forming bacteria tend to cause chronic infections whereby infections persist despite seemingly adequate antibiotic therapy. This review briefly describes the responses of biofilm matrix components and biofilm-associated bacteria towards sub-lethal concentrations of antimicrobial agents, which may include the generation of genetic and phenotypic variabilities. Clinical implications of bacterial biofilms in relation to antibiotic treatments are also discussed.

Short report: isolation of Escherichia coli O157:H7 from fecal samples of cows in Vietnam.

Enterohemorrhagic Escherichia coli O157:H7 was isolated for the first time in Vietnam. Shiga toxin-producing E. coli were isolated from 8 of 100 cows examined. The two strains showing serotype O157:H7 carried the eae, ehxA, and stx2c genes, but the other six were negative for the eae gene.

Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method.

Here we report a loop-mediated isothermal amplification (LAMP) method for detecting Shigella and enteroinvasive Escherichia coli. The target for this LAMP method is the ipaH gene which is carried by both of the pathogens. The LAMP method efficiently detected the gene within 2 h at a minimal amount of bacteria (8 CFU) per reaction.

Pathogenicity of Shigella in healthy carriers: a study in Vientiane, Lao People's Democratic Republic.

Shigella spp. isolated from diarrheal patients and non-diarrheal carriers were examined by PCR for the presence of two pathogenic genes, chromosomal ipaH and invasive plasmid encoded ial. Shigella spp. were detected in 7 of 72 diarrheal cases examined (9.7%), and 9 of 145 non-diarrheal cases (6.2%). All isolates from diarrheal cases harbored both ipaH and ial, while all isolates from non-diarrheal cases were positive for ipaH but not ial. These results suggested that Shigella spp. in healthy carriers were basically non-pathogenic.

Purification and characterization of a novel metalloprotease isolated from Aeromonas caviae.

A novel protease produced by Aeromonas caviae was purified and characterized. The molecular weight of the protease (AP19) was estimated as 19 kDa on SDS-polyacrylamide gel electrophoresis. The protease activity was not inhibited completely by heating at 100 degrees C for 15 min. The proteolytic activities were inhibited by metalloprotease inhibitor. The N-terminal amino acid sequence of AP19 was VTASVSFSGRCTN. AP19 did not activate Aeromonas proaerolysin, and did not show fluid accumulation in the rabbit intestinal loop test. A high concentration of the protease showed cytotoxic activity against Vero cells.

Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria.

Aeromonas veronii is an opportunistic human pathogen that causes diarrhoea and extraintestinal infections, such as wound infection and septicaemia. An A. veronii protease (AVP) from a biovar sobria strain, AeG1, was partially purified and characterized. Mature AVP hydrolysed casein but not elastin, and protease activity was inhibited by metalloprotease inhibitors. Nucleotide sequence analysis showed that AVP belongs to the thermolysin family of proteases. An AVP-deficient mutant was constructed to investigate the role of AVP in aerolysin activation. Western blot analysis using anti-aerolysin antisera revealed that proaerolysin (52 kDa) in the AVP-deficient mutant was not completely activated to mature aerolysin (47 kDa) as seen in the wild-type strain. The AVP-deficient mutant showed lower cytotoxic and haemolytic activities than wild type. AVP and proaerolysin had no haemolytic activity; however, activity appeared after incubating both proteins. Taken together, these results suggested that AVP plays an indirect role in virulence through activating aerolysin, which is an essential step for cytotoxic activity.

Vibrio cholerae utilizes direct sRNA regulation in expression of a biofilm matrix protein.

Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor σE. In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the σE-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.

VrrA mediates Hfq-dependent regulation of OmpT synthesis in Vibrio cholerae.

OmpT, an outer membrane porin of Vibrio cholerae, is tightly regulated by the organism in response to different environments. Two transcriptional regulators, cAMP receptor protein (CRP) and ToxR, compete at the ompT promoter region. CRP activates ompT transcription by a loop-forming mechanism, while ToxR functions as an antiactivator and repressor, depending on its interplay with CRP. VrrA, a 140-nt small noncoding RNA in V. cholerae, is controlled by the alternative sigma factor sigma(E). We have demonstrated previously that VrrA represses ompA translation by base-pairing with the 5' region of the mRNA, thereby affecting the release of outer membrane vesicles and modulating the colonization ability of V. cholerae. In this study, we demonstrate that VrrA RNA represses ompT translation by base-pairing with the 5' region of the mRNA and that regulation requires the RNA chaperone protein Hfq. These results add new insight into the regulation of OmpT. In addition to pH/temperature signals via the ToxR regulon and carbon source signals via the cAMP-CRP complex, OmpT is further regulated by signals received via the sigma(E) regulon through VrrA.

A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing.

Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.

Enteroaggregative Escherichia coli: incidence in Japan and usefulness of the clump-formation test.

The usefulness of the clump-formation test described by Albert et al. for identifying enteroaggregative Escherichia coli (EAggEC) and the incidence of EAggEC in Japan were studied. One hundred and seventy strains of E. coli agglutinated with enteropathogenic E. coli diagnostic antisera were collected from a variety of districts in Japan. All isolates were from diarrheal stools. EAggEC was identified on the basis of the presence of the aggR gene accompanied by aggregative adhesion to HEp-2 cells. After 24 strains carrying eaeA, elt, est, stx-1, stx-2, or ipaH genes were eliminated, the remaining 145 strains were examined for adhesion to Hep-2 cells, the presence of the aggRgene, and clump formation on the surface of Muller-Hinton broth. aggR was detected in 10 strains, and 9 of them displayed aggregative adhesion to HEp-2 cells. Seven strains produced marked clumps and 22 showed moderate clump formation. The sensitivity and specificity of the clump-formation test for detecting EAggEC were each about 90%, and they varied slightly depending on the stringency of evaluation for the degree of clump formation. From these results, we conclude that the incidence of EAggEC cannot be ignored as a possible cause of diarrheal disease in Japan, and we strongly recommend the clump-formation test for detecting EAggEC.

Distribution of putative adhesins in different seropathotypes of Shiga toxin-producing Escherichia coli.

The distribution of eight putative adhesins that are not encoded in the locus for enterocyte effacement (LEE) in 139 Shiga toxin-producing Escherichia coli (STEC) of different serotypes was investigated by PCR. Five of the adhesins (Iha, Efa1, LPF(O157/OI-141), LPF(O157/OI-154), and LPF(O113)) are encoded in regions corresponding to genomic O islands of E. coli EDL933, while the other three adhesins have been reported to be encoded in the STEC megaplasmid of various serotypes (ToxB [O157:H7], Saa [O113:H21], and Sfp [O157:NM]). STEC strains were isolated from humans (n = 54), animals (n = 52), and food (n = 33). They were classified into five seropathotypes (A through E) based on the reported occurrence of STEC serotypes in human disease, in outbreaks, and in the hemolytic-uremic syndrome (M. A. Karmali, M. Mascarenhas, S. Shen, K. Ziebell, S. Johnson, R. Reid-Smith, J. Isaac-Renton, C. Clark, K. Rahn, and J. B. Kaper, J. Clin. Microbiol. 41:4930-4940, 2003). The most prevalent adhesin was that encoded by the iha gene (91%; 127 of 139 strains), which was distributed in all seropathotypes. toxB and efa1 were present mainly in strains of seropathotypes A and B, which were LEE positive. saa was present only in strains of seropathotypes C, D, and E, which were LEE negative. Two fimbrial genes, lpfA(O157/OI-141) and lpfA(O157/OI-154), were strongly associated with seropathotype A. The fimbrial gene lpfA(O113) was present in all seropathotypes except for seropathotype A, while sfpA was not present in any of the strains studied. The distribution of STEC adhesins depends mainly on serotypes and not on the source of isolation. Seropathotype A, which is associated with severe disease and frequently is involved in outbreaks, possesses a unique adhesin profile which is not present in the other seropathotypes. The wide distribution of iha in STEC strains suggested that it could be a candidate for vaccine development.