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Regulation of Virulence Genes by Quorum and Iron in Vibrio vulnificus

  • 김인황 (In Hwang Kim, 생명과학과 미생물학 전공)

초록/요약 도움말

The gene vvpE, encoding the virulence factor elastase, is a member of the quorum-sensing regulon in Vibrio vulnificus and displays enhanced expression at high cell density. We observed that this gene was repressed under iron-rich conditions and that the repression was due to a Fur (Ferric uptake regulator)-dependent repression of smcR, a gene encoding a quorum-sensing master regulator with similarity to luxR in V. harveyi. A gel mobility shift assay and a footprinting experiment demonstrated that the Fur-iron complex binds directly to two regions upstream of smcR (-82 to -36 and -2 to +27, with respect to the transcription start site) with different affinities. However, binding of the Fur-iron complex is reversible enough to allow expression of smcR to be induced by quorum sensing at high cell density under iron-rich conditions. Under iron-limiting conditions, Fur fails to bind either region and the expression of smcR is regulated solely by quorum sensing. These results suggest that two biologically important environmental signals, iron and quorum sensing, converge to direct the expression of smcR, which then coordinates the expression of virulence factors. In contrast to that seen for SmcR of V. vulnificus, expression of the quorum-sensing master regulator HapR in wild-type V. cholerae was not affected by iron. However, in a fur-deletion mutant of V. cholerae, the expression of hapR was higher than in wild-type in the absence of iron and further enhanced in the presence of iron.

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초록/요약 도움말

Vibrio vulnificus is an opportunistic pathogen, which causes a septicemia in humans. Using a DNA microarray containing 131 genes possibly associated with pathogenicity, transport, signal transduction, and gene regulations in the pathogen, the gene expression among four clinical and six environmental V. vulnificus isolates was compared. cDNAs from total RNAs of these isolates were hybridized into the DNA microarray chip using the cDNA of the wild-type MO6-24/O strain as a reference. We focused on selecting differentially expressed (DE) genes between clinical and environmental isolates using a modified t-statistic. Two R pacakges, limma and multtest, employed to adjust for the multiple testing in a microarray, and then listed the top ten genes in terms of the adjusted p-values. Two statistically significant DE genes between virulent isolates and less-virulent isolates with a marginal statistical significance were detected. These genes were putatively encoding pilin and adenlyate cylase. Real Time-PCR confirmed that these two selected genes transcribed in significantly higher levels in virulent isolates than in less-virulent isolates. And one gene, VVMO6_00510, was also identified at our previously In-Vivo-Expression-Technology (IVET) analysis. A protein possibly encoded by the gene has the inverzincin motif (HXXEH), a feature common in other insulin-degrading enzymes. Recombinant proteins encoded by the gene exhibited insulin-degrading activity. The protein possesses the N-terminal signal peptide, and was detected mainly in supernatant, suggesting that the enzyme is an extracellular protein. As such this gene was named SidC (Secreted insulin degrading enzyme C), which, when combined with two other genes, named sidA and sidB, respectively, forms an operon. When comparing mice infected by the wild-type V. vulnificus MO6-24/O strain, it was found that mice infected by the sidC-deleted isogenic mutant showed a lower level of survival ability and a higher level of blood insulin. Furthermore, compared to the wild-type, the sidC mutant displayed attenuated virulence and a delayed proliferation in the mouse model. These results suggest that SidC facilitates the survival and proliferation of the pathogen in host.

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