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Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence.

ABSTRACT: Atlantic salmon (Salmo salar) were challenged with Vibrio anguillarum strains M93Sm and NB10 and empA null mutants M99 and NB12. Both wild types were virulent when administered by intraperitoneal (i.p.) injection or anal intubation. NB12 was avirulent via either route of infection. M99 virulence was attenuated when delivered by intubation, but fully virulent by i.p. injection. Northern blot analysis revealed empA expression in M93Sm and NB10 cells incubated in mucus, while incubation in Luria-Bertani broth plus 2% NaCl (LB20) induced empA expression only in NB10. Nucleotide differences between M93Sm and NB10 empA sequences were found in regions located 207 and 229 bp upstream of the empA translational start. Reverse transcription-PCR and 5' rapid amplification of cDNA ends revealed the empA transcriptional start site 85 bp upstream of the translational start for both strains. A putative sigma(S)-dependent promoter was identified upstream of the transcriptional start in both strains. Site-directed mutagenesis was used to create rpoS mutants of M93Sm and NB10. Neither rpoS mutant exhibited protease activity. Since empA is expressed during stationary phase, the effects of conditioned medium on protease activity were examined. M99 conditioned LB20 supernatants stimulated protease activity in NB10 while allowing M93Sm to produce protease in LB20. Neither acyl homoserine lactones nor AI-2 induced protease activity. Conditioned LB20 supernatant from a V. anguillarum luxS mutant caused a more rapid induction of protease activity in wild-type cells. Our data show that expression of empA is differentially regulated in V. anguillarum strains NB10 and M93Sm and requires sigma(S), quorum-sensing molecules, and gastrointestinal mucus.

SUBMITTER: Denkin SM

PROVIDER: S-EPMC444792 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence.

Denkin Steven M SM Nelson David R DR

Applied and environmental microbiology 20040701 7

Atlantic salmon (Salmo salar) were challenged with Vibrio anguillarum strains M93Sm and NB10 and empA null mutants M99 and NB12. Both wild types were virulent when administered by intraperitoneal (i.p.) injection or anal intubation. NB12 was avirulent via either route of infection. M99 virulence was attenuated when delivered by intubation, but fully virulent by i.p. injection. Northern blot analysis revealed empA expression in M93Sm and NB10 cells incubated in mucus, while incubation in Luria-Be ...[more]

PMID: 15240301

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Project description:BACKGROUND: The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements. RESULTS: Two strains of V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 microg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer. CONCLUSIONS: The data demonstrate that protein extracts of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm.

Project description:Vibrio anguillarum causes a hemorrhagic septicemia that affects cold- and warm-water adapted fish species. The main goal of this work was to determine the temperature-dependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25°C, the degree of virulence of V. anguillarum RV22 is higher at 15°C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15°C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm- or cold-water temperatures. The comparative analysis of transcriptomes at high- and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25°C than at 15°C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15°C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25°C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

Dataset Information

Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence.

Publications

Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence.

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