HlyF Produced by Extraintestinal Pathogenic Escherichia coli Is a Virulence Factor That Regulates Outer Membrane Vesicle Biogenesis.

Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.

Molecular analysis of dominant species in Listeria monocytogenes-positive biofilms in the drains of food processing facilities.

Listeria monocytogenes exhibits symbiotic codependence with the dominant commensal bacteria, which may help it avoid being removed or inactivated by disinfectants in local environments. In this study, we investigated L. monocytogenes-positive biofilms at food production facilities, and the dominant bacterial species of the biofilms were identified to determine the properties of the microbiological background. For this purpose, the ISO 11290 method was used for the detection and isolation of L. monocytogenes, and the species were further identified based on 16S rRNA and hly genes. 16S rRNA gene-based cloning, terminal restriction fragment length polymorphism, and denaturing gradient gel electrophoresis were combined to evaluate the dominant bacteria of the drain biofilms. Out of 100 drain samples, 8 were naturally contaminated with L. monocytogenes. Three molecular methods consistently showed that Pseudomonas psychrophila, Pseudomonas sp., and Klebsiella oxytoca were dominant species in 3Q, 5Q, and 6Q samples; Aeromonas hydrophila and Klebsiella sp. were significantly dominant in 1-2, 1-3, and 3-2 samples; A. hydrophila and K. oxytoca were dominant in the 2-3 sample; and A. hydrophila and Pseudomonas sp. were prominent in the 3-3 sample. Different biofilms from the same plant shared common bands, suggesting that similar bacteria can be found and can be dominant in different biofilms. This study provides a better understanding of the dominant compositions in these bacterial communities. Further studies to determine the mechanism of co-culture with L. monocytogenes will be of critical importance in predicting effective disinfection strategies.

The regulator HlyU, the repeat-in-toxin gene rtxA1, and their roles in the pathogenesis of Vibrio vulnificus infections.

HlyU is a master regulator that plays an essential role in the virulence of the human pathogen Vibrio vulnificus. One of the most noteworthy characteristics of HlyU regulation in this organism is its positive control of the expression of the repeat-in-toxin (RtxA1) gene, one of the most important virulence factors accounting for the fulminating and damaging nature of V. vulnificus infections. In this work, we reviewed the latest studies of RtxA1 in this bacterium and highlight the mechanism of gene regulation of rtxA1 expression by HlyU under a broader gene regulatory network.

Crystal structure of the transcriptional activator HlyU from Vibrio vulnificus CMCP6.

HlyU is a transcription factor of the ArsR/SmtB family and activates the expression of the pathogenic Vibrio vulnificus RTX toxin. In contrast to the other metal-responding ArsR/SmtB proteins, HlyU does not sense metal ions. To provide its structural information, we elucidated the crystal structure of HlyU from V. vulnificus CMCP6 (HlyU_Vv). The monomeric HlyU_Vv architecture of five alpha-helices and two beta-strands, some of which constitute a typical DNA-binding winged helix-turn-helix (wHTH) motif, is very similar to that of other transcription regulators. Nonetheless, the homo-dimeric HlyU_Vv structure shows several different, three-dimensional features in the spatial position and the detailed dimeric interaction, which were not observed in the modeling study based on the same protein family and sequence similarity.

[Influence of Lactobacillus fermentum metabolites on ultrastructure of pathogenic Escherichia coli].

AIM: To study ultrastructural changes of hemolytic and enterohemorrhagic Escherichia coli during interaction with metabolites of Lactobacillus fermentum. MATERIALS AND METHODS: Strains of pathogenic hemolytic (Hly) and enterohemorrhagic Escherichia coli (O157:H7) as well as symbiotic bacteriocinogenic strain Lactobacillus fermentum 97 were used. Inhibition of growth of viable cells was performed by delayed antagonism method. Using electron microscopy, assessment of ultrastructural changes of hemolytic and enterohemorrhagic E. coli under the influence of diffusing in MPC-agar metabolites of lactobacilli. RESULTS: Changes pointing to profound destructive processes in bacterial cells were detected on ultrathin sections. Under the influence of diffusing metabolites of lactobacilli, the following changes were observed: destabilization of cell wall, expansion of periplasmatic space, and emergence of low electron density areas of cytoplasm in polar sections of cells with visualization of floccular material. Emergence of elongated paracrystallic packings and filamentous structures of different length, which deserve special study, was observed in cells of hemolytic E. coli. CONCLUSION: Bacteriocin-like products of lactobacilli during interaction with pathogenic E. coli cause profound destructive changes in the latter which lead to destruction of target cells.

Regulation of the Enterococcus faecalis pAD1-related sex pheromone response: analyses of traD expression and its role in controlling conjugation functions.

The Enterococcus faecalis haemolysin plasmid pAD1 (60 kb) confers a conjugative mating response to an octapeptide sex pheromone (cAD1) secreted by plasmid-free strains. The response involves two plasmid-borne regulatory determinants: traE1, whose product positively regulates all or most conjugation-related structural genes; and traA, whose product negatively regulates traE1 by controlling transcriptional readthrough of an upstream termination site (TTS1/TTS2). TraA binds to the promoter region of iad, which encodes a pheromone-inhibitor peptide, iAD1; and TTS1/TTS2 tightly terminates transcription arriving from this promoter during the uninduced state. A determinant, traD, appearing to encode a small peptide (23 amino acids), is located just downstream of iad and is in the opposite orientation. Transcripts of traD were identified and found to be present at a relatively high level in cells not expressing conjugation functions; the amount of RNA was greatly reduced, however, upon induction of the pheromone response. The decrease in traD RNA was not a consequence of the induced activity of TraE1, as it also occurred in a traE1 insertion mutant. A mutation in traD that would eliminate translation but that did not affect transcription had no apparent effect on the cell phenotype, indicating that RNA was likely to be the functional product. This was consistent with our finding that synthesis of traD RNA containing the translational defect was able to complement, in trans, a temperature-sensitive traD mutation. Thus, transcription of the traD determinant is significantly involved in downregulation of the pAD1 pheromone response.

A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence.

PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence.