Expression and Quorum Sensing Regulation of Type III Secretion System Genes of Vibrio harveyi during Infection of Gnotobiotic Brine Shrimp.

Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells

Quorum sensing regulation of virulence gene expression in Vibrio harveyi during its interaction with marine diatom Skeletonema marinoi

Communication between species from different kingdoms may be as important as intra-kingdom communication. It has recently been confirmed that co-existing bacteria and phytoplankton in aquatic ecosystems do cross-talk. This study examined the signs of possible cross signalling between V. harveyi, one of the predominant bacterial species of the marine ecosystem and a dominant diatom species, S.marinoi, to understand communication over species borders. It is known that V.harveyi employ quorum sensing for cell-to-cell communication, bioluminescence (luxR), and the regulation of the virulence gene (vhp, chiA). Former studies have also shown, this kind of interactions being disrupted by compounds secreted by a few algal species existing in the aquatic ecosystem. We investigated the QS communication by quantifying the expression levels of virulence regulator luxR and virulence factors metalloprotease (vhp) and chitinase (chiA) in four different V. harveyi strains grown in the presence of S. marinoi strain. Results obtained in this study indicate that quorum sensing was activated in strains of V. harveyi analysed but did not regulate the expressions of vhp and chiA virulence factors. This observation suggests that the existence of S. marinoi did not interfere with the QS behavior of V. harveyi and its interaction with marine diatom; it may be due to the commensalism relationship

<i>In vivo</i> expression of the type III secretion system genes <i>vopD</i>, <i>vcrD</i> and <i>vscP</i>.

<p><i>In vivo</i> expression of the type III secretion system genes <i>vopD</i> <b>(A)</b>, <i>vcrD</i> <b>(B)</b> and <i>vscP</i> <b>(C)</b> in wild type <i>V</i>. <i>harveyi</i> (WT) and mutants with the quorum sensing system locked in high cell density configuration (QS^c) and the quorum sensing system locked in low cell density configuration (QS^-), respectively, during infection of brine shrimp larvae. The expression in wild type <i>V</i>. <i>harveyi</i> at the 0.5h time point was set at 1 and the expression in all strains at all time points was normalised accordingly using the 2^-ΔΔCT method. The error bars represent the standard deviation of three independent shrimp cultures (each time based on bacterial mRNAs extracted from 500 larvae). The RNA polymerase A subunit (<i>rpoA</i>) mRNA was used to normalise between strains.</p

Expression and Quorum Sensing Regulation of Type III Secretion System Genes of <i>Vibrio harveyi</i> during Infection of Gnotobiotic Brine Shrimp

<div><p>Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of <i>Vibrio harveyi</i>, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in <i>V</i>. <i>harveyi</i> increased within the first 12h after challenge and decreased again thereafter. The <i>in vivo</i> expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, <i>in vivo</i> expression levels of the type III secretion system genes were much (> 1000 fold) higher than the <i>in vitro</i> expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells.</p></div

Quorum sensing in <i>Vibrio harveyi</i>.

<p>The LuxM, LuxS and CqsA enzymes synthesise the signal molecules HAI-1, AI-2 and CAI-1, respectively. These signal molecules are detected at the cell surface by the LuxN, LuxQ and CqsS two-component receptor proteins, respectively. Detection of AI-2 by LuxQ requires the periplasmic protein LuxP. <b>(A)</b> In the absence of signal molecules, the receptors autophosphorylate and transfer phosphate to LuxO via LuxU. Phosphorylation activates LuxO, which together with σ⁵⁴ activates the production of five small regulatory RNAs (sRNAs). These sRNAs, together with the chaperone Hfq, destabilise the mRNA encoding the transcriptional regulator LuxR. Therefore, in the absence of autoinducers, the LuxR protein is not produced. LuxR is a repressor of ExsA. Hence, in the absence of signal molecules, ExsA is produced and in turn activates expression of the TTSS operons. <b>(B)</b> In the presence of high concentrations of the signal molecules, the receptor proteins switch from kinases to phosphatases, which results in dephosphorylation of LuxO. Dephosphorylated LuxO is inactive and therefore, the sRNAs are not formed and the transcriptional regulator LuxR is produced. LuxR represses ExsA, and the TTSS operons are not expressed. “P” denotes phosphotransfer.</p