In Hwang Kim

Cyclo(Phe-Pro) Modulates the Expression of ompU in Vibrio spp.

Vibrio vulnificus was found to produce a chemical that induced the expression of Vibrio fischeri lux genes. Electron spray ionization-mass spectrometry and 1H nuclear magnetic resonance analyses indicated that the compound was cyclo(L-Phe-L-Pro) (cFP). The compound was produced at a maximal level when cell cultures reached the onset of stationary phase. Sodium dodecyl sulfate-polyacrylamide gel analysis of the total proteins of V. vulnificus indicated that expression of OmpU was enhanced by exogenously added synthetic or purified cFP. A toxR-null mutant failed to express ompU despite the addition of cFP. The related Vibrio spp. V. cholerae, V. parahaemolyticus, and V. harveyi also produced cFP, which induced the expression of their own ompU genes. cFP also enhanced the expression in V. cholerae of the ctx genes, which are known to be regulated by ToxR. Our results suggest that cFP is a signal molecule controlling the expression of genes important for the pathogenicity of Vibrio spp.

The Fur-Iron Complex Modulates Expression of the Quorum-Sensing Master Regulator, SmcR, To Control Expression of Virulence Factors in Vibrio vulnificus

ABSTRACT 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 Vibrio 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 differing 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.

Iron and Quorum Sensing Coordinately Regulate the Expression of Vulnibactin Biosynthesis in Vibrio vulnificus

Background: vvsAB are required for the vulnibactin biosynthesis important for the virulence of V. vulnificus. Results: Fur and SmcR regulate vvsA expression by differential binding to vvsA promoter region in response to iron concentration. Conclusion: Fur and SmcR regulate vulnibactin synthesis in V. vulnificus, ensuring an appropriate intracellular iron concentration. Significance: Characterizing the vulnibactin biosynthesis regulation mediated by Fur and SmcR is important to understanding the bacterial pathogenesis. Vibrio vulnificus is a halophilic marine pathogen associated with human diseases such as septicemia and serious wound infections. Genes vvsA and vvsB, which are co-transcribed and encode a member of the nonribosomal peptide synthase family, are required for vulnibactin biosynthesis in V. vulnificus. In this study, we found that quorum sensing represses the transcription of a vvsAB-lux reporter fusion. Gel shift assay and DNaseI footprinting experiments show that the main regulator of quorum sensing, SmcR, binds to a 22-bp region located between −40 and −19 with respect to the vvsA transcription start site. Mutation of the SmcR binding site abolishes the repression of vvsA::luxAB by SmcR. Fur represses vvsAB transcription in the presence of iron by binding to a 47-bp region located between −45 and +2 with respect to the vvsA transcription start site. A competition gel shift assay and footprinting experiment using Fur and SmcR showed that Fur binds to the vvsA promoter region with higher affinity than SmcR. Studies with the vvsAB::luxAB transcriptional fusion demonstrate that in the presence of iron, Fur is the key repressor of vvsAB transcription, whereas in iron-limited conditions, SmcR is the key regulator repressing vvsAB transcription. This study demonstrates that the Fe-Fur complex and quorum sensing cooperate to repress the transcription of vvsAB in response to iron conditions, suggesting that fine tuning of the intracellular iron level is important for the survival and pathogenicity of V. vulnificus.

Iron- and Quorum-sensing Signals Converge on Small Quorum-regulatory RNAs for Coordinated Regulation of Virulence Factors in Vibrio vulnificus

Vibrio vulnificus is a marine bacterium that causes human infections resulting in high mortality. This pathogen harbors five quorum-regulatory RNAs (Qrr1–5) that affect the expression of pathogenicity genes by modulating the expression of the master regulator SmcR. The qrr genes are activated by phosphorylated LuxO to different degrees; qrr2 is strongly activated; qrr3 and qrr5 are moderately activated, and qrr1 and qrr4 are marginally activated and are the only two that do not respond to cell density-dependent regulation. Qrrs function redundantly to inhibit SmcR at low cell density and fully repress when all five are activated. In this study, we found that iron inhibits qrr expression in three distinct ways. First, the iron-ferric uptake regulator (Fur) complex directly binds to qrr promoter regions, inhibiting LuxO activation by competing with LuxO for cis-acting DNA elements. Second, qrr transcription is repressed by iron independently of Fur. Third, LuxO expression is repressed by iron independently of Fur. We also found that, under iron-limiting conditions, the five Qrrs functioned additively, not redundantly, to repress SmcR, suggesting that cells lacking iron enter a high cell density mode earlier and could thereby modulate expression of virulence factors sooner. This study suggests that iron and quorum sensing, along with their cognate regulatory circuits, are linked together in the coordinated expression of virulence factors.

Cyclo(Phe-Pro) Produced by the Human Pathogen Vibrio vulnificus Inhibits Host Innate Immune Responses through the NF-κB Pathway

ABSTRACT Cyclo(Phe-Pro) (cFP) is a secondary metabolite produced by certain bacteria and fungi. Although recent studies highlight the role of cFP in cell-to-cell communication by bacteria, its role in the context of the host immune response is poorly understood. In this study, we investigated the role of cFP produced by the human pathogen Vibrio vulnificus in the modulation of innate immune responses toward the pathogen. cFP suppressed the production of proinflammatory cytokines, nitric oxide, and reactive oxygen species in a lipopolysaccharide (LPS)-stimulated monocyte/macrophage cell line and in bone marrow-derived macrophages. Specifically, cFP inhibited inhibitory κB (IκB) kinase (IKK) phosphorylation, IκBα degradation, and nuclear factor κB (NF-κB) translocation to the cell nucleus, indicating that cFP affects the NF-κB pathway. We searched for genes that are responsible for cFP production in V. vulnificus and identified VVMO6_03017 as a causative gene. A deletion of VVMO6_03017 diminished cFP production and decreased virulence in subcutaneously inoculated mice. In summary, cFP produced by V. vulnificus actively suppresses the innate immune responses of the host, thereby facilitating its survival and propagation in the host environment.

Cyclo(phenylalanine-proline) induces DNA damage in mammalian cells via reactive oxygen species

Cyclo(phenylalanine‐proline) is produced by various organisms such as animals, plants, bacteria and fungi. It has diverse biological functions including anti‐fungal activity, anti‐bacterial activity and molecular signalling. However, a few studies have demonstrated the effect of cyclo(phenylalanine‐proline) on the mammalian cellular processes, such as cell growth and apoptosis. In this study, we investigated whether cyclo(phenylalanine‐proline) affects cellular responses associated with DNA damage in mammalian cells. We found that treatment of 1 mM cyclo(phenylalanine‐proline) induces phosphorylation of H2AX (S139) through ATM‐CHK2 activation as well as DNA double strand breaks. Gene expression analysis revealed that a subset of genes related to regulation of reactive oxygen species (ROS) scavenging and production is suppressed by the cyclo(phenylalanine‐proline) treatment. We also found that cyclo(phenylalanine‐proline) treatment induces perturbation of the mitochondrial membrane, resulting in increased ROS, especially superoxide, production. Collectively, our study suggests that cyclo(phenylalanine‐proline) treatment induces DNA damage via elevation of ROS in mammalian cells. Our findings may help explain the mechanism underlying the bacterial infection‐induced activation of DNA damage response in host mammalian cells.

Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo

Background: Vibrio vulnficus produces SidC, an extracellular insulin-degrading enzyme. Results: SidC causes degradation of insulin, leading to proliferation of the pathogen, and sidC was expressed in low-glucose conditions. Conclusion: Degradation of insulin by SidC correlated with the proliferation of the pathogen. Significance: V. vulnificus manipulates host endocrine signals through SidC, making the host environment more favorable for its own proliferation. We describe a novel insulin-degrading enzyme, SidC, that contributes to the proliferation of the human bacterial pathogen Vibrio vulnificus in a mouse model. SidC is phylogenetically distinct from other known insulin-degrading enzymes and is expressed and secreted specifically during host infection. Purified SidC causes a significant decrease in serum insulin levels and an increase in blood glucose levels in mice. A comparison of mice infected with wild type V. vulnificus or an isogenic sidC-deletion strain showed that wild type bacteria proliferated to higher levels. Additionally, hyperglycemia leads to increased proliferation of V. vulnificus in diabetic mice. Consistent with these observations, the sid operon was up-regulated in response to low glucose levels through binding of the cAMP-receptor protein (CRP) complex to a region upstream of the operon. We conclude that glucose levels are important for the survival of V. vulnificus in the host, and that this pathogen uses SidC to actively manipulate host endocrine signals, making the host environment more favorable for bacterial survival and growth.