The LuxR family protein SpnR functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in Serratia marcescens

Serratia marcescens SS-1 produces at least four N-acylhomoserine lactones (AHLs) which were identified using high-resolution mass spectrometry and chemical synthesis, as N-(3-oxohexanoyl) homo-serine lactone (3-oxo-C6-HSL), N-hexanoyl- (C6-HSL), N-heptanoyl (C7-HSL) and N-octanoyl- (C8-HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation. spnR overexpression and spnR gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator. spnI overexpression, the provision of exogenous AHLs and spnI gene deletion suggest that SpnR is de-repressed by 3-oxo-C6-HSL. In addition, long chain AHLs antagonize the biosurfactant-mediated surface translocation of S. marcescens SS-1. Upstream of spnI there is a gene which we have termed spnT. spnI and spnT form an operon and although database searches failed to reveal any spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.     

Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria

Changes in DNA supercoiling in response to environmental signals such as osmolarity, temperature, or anaerobicity appear to play an underlying role in the regulation of gene expression in bacteria. Extensive genetic analyses have implicated the osmZ gene in this regulatory process: osmZ mutations are highly pleiotropic and alter the topology of cellular DNA. We have shown that the product of the osmZ gene is the "histone-like" protein H1 (H-NS). Protein H1 is one of the most abundant components of bacterial chromatin and binds to DNA in a relatively nonspecific fashion. These data imply a regulatory role for one of the major components of bacterial chromatin and provide support for the notion that changes in DNA topology and/or chromatin structure play a role in regulating gene expression.     

Synthetic homoserine lactone-derived sulfonylureas as inhibitors of Vibrio fischeri quorum sensing regulator

A series of 9 homoserine lactone-derived sulfonylureas substituted by an alkyl chain, some of them bearing a phenyl group at the extremity, have been prepared. All compounds were found to inhibit the action of 3-oxo-hexanoyl-L-homoserine lactone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri, the aliphatic compounds being more active than their phenyl-substituted counterparts. Molecular modelling studies performed on the most active compound in each series suggest that the antagonist activity could be related to the perturbation of the hydrogen-bond network in the ligand-protein complexes.     

Detection of quorum sensing systems of bacteria isolated from fouled marine organisms

N-Acyl homoserine lactones (AHLs or N-AHLs) are a class of signaling molecules involved in bacterial quorum sensing (qs) that have recently been proposed as mediators of the fouling process. In this study, we determined the presence of AHLs in the following marine bacteria strains, which were collected in Santa Marta Bay (Colombia) from heavily fouled surfaces: Ochrobactrum sp., Vibrio sp. (23-6PIN), Vibrio campbellii, Vibrio sp. (11-6DEP), Ochrobactrum pseudogringnonense, Shewanella sp., Vibrio harveyi and Alteromonas sp. The detection and identification of AHLs was conducted using the microbial biosensor Escherichia coli (pSB401) and GC–MS and HPLC-MS analyses. We found that all isolated marine strains had quorum sensing systems mediated by either N-butanoyl homoserine lactone or N-hexanoyl homoserine lactone and in some cases by both. These results are in agreement with the theory that qs is involved in the fouling process. It is noteworthy to mention that we identified qs systems for the first time in bacteria of the genera Ochrobactrum and Alteromonas.     

SLAM-associated protein as a potential negative regulator in Trk signaling

Neurotrophin signaling plays important roles in regulating the survival, differentiation, and maintenance of neurons in the nervous system. Binding of neurotrophins to their cognate receptors Trks induces transactivation and phosphorylation of the receptor at several tyrosine residues. These phosphorylated tyrosine residues then serve as crucial docking sites for adaptor proteins containing a Src homology 2 or phosphotyrosine binding domain, which upon association with the receptor initiates multiple signaling events to mediate the action of neurotrophins. Here we report the identification of a Src homology 2 domain-containing molecule, SLAM-associated protein (SAP), as an interacting protein of TrkB in a yeast two-hybrid screen. SAP was initially identified as an adaptor molecule in SLAM family receptor signaling for regulating interferon-gamma secretion. In the current study, we found that SAP interacted with TrkA, TrkB, and TrkC receptors in vitro and in vivo. Binding of SAP required Trk receptor activation and phosphorylation at the tyrosine 674 residue, which is located in the activation loop of the kinase domain. Overexpression of SAP with Trk attenuated tyrosine phosphorylation of the receptors and reduced the binding of SH2B and Shc to TrkB. Moreover, overexpression of SAP in PC12 cells suppressed the nerve growth factor-dependent activation of extracellular signal-regulated kinases 1/2 and phospholipase Cgamma, in addition to inhibiting neurite outgrowth. In summary, our findings demonstrated that SAP may serve as a negative regulator of Trk receptor activation and downstream signaling.     

Antibody catalyzed hydrolysis of a quorum sensing signal found in Gram-negative bacteria

N-(3-Oxo-acyl) homoserine lactones are used by Gram-negative bacteria to signal the establishment of specific population densities and coordinate population-wide gene expression. Herein we report the antibody-catalyzed hydrolysis of N-(3-oxo-acyl) homoserine lactone (AHL) using a reactive immunization strategy with a squaric monoester monoamide hapten. Kinetic analysis of the most efficient antibody revealed a modest k(cat), with AHL hydrolysis competitively inhibited by original squaric monoester monoamide hapten. These studies suggest that antibody catalysis could provide a new avenue for blocking quorum sensing in bacteria.     

青岛近海沉积物生物被膜中密度感应及密度感应淬灭细菌多样性

【背景】细菌密度感应(Quorum sensing,QS)是指细菌利用分泌的信号分子进行相互交流的现象,而密度感应淬灭(Quorumquenching,QQ)是指通过干扰信号分子的产生、释放、积累或应答从而阻抑密度感应通路。【目的】探究青岛近海沉积物生物被膜中密度感应和密度感应淬灭细菌的多样性。【方法】采用海水培养基2216E从青岛近海沉积物生物被膜中分离获取可培养细菌,采用平板交互划线和高通量筛选的方法分别筛选具有密度感应和密度感应淬灭的菌株。【结果】共分离获得83株共54种具有密度感应和密度感应淬灭的细菌,分属于四大细菌门类:变形菌门、拟杆菌门、厚壁菌门和放线菌门。其中,38株(45.8%)可以产生酰基高丝氨酸内酯(Acyl-homoserine lactone,AHL)类信号分子,它们分属于变形菌门(37株,15种)和拟杆菌门(1株,1种),优势属为弧菌属和鲁杰氏菌属;能够降解AHL类信号分子的有57株(68.7%),在变形菌门(41株,23种)、拟杆菌门(14株,10种)、厚壁菌门(5株,5种)以及放线菌门(1株,1种)中均有分布。【结论】在青岛近海沉积物生物被膜可培养细菌中,具有密度感应和密度感应淬灭现象的菌株具有很高的丰度和多样性,为后续生态学意义的研究与海洋微生物的开发提供了参考。     

Food as a source for quorum sensing inhibitors: iberin from horseradish revealed as a quorum sensing inhibitor of Pseudomonas aeruginosa

Foods with health-promoting effects beyond nutritional values have been gaining increasing research focus in recent years, although not much has been published on this subject in relation to bacterial infections. With respect to treatment, a novel antimicrobial strategy, which is expected to transcend problems with selective pressures for antibiotic resistance, is to interrupt bacterial communication, also known as quorum sensing (QS), by means of signal antagonists, the so-called QS inhibitors (QSIs). Furthermore, QSI agents offer a potential solution to the deficiencies associated with use of traditional antibiotics to treat infections caused by bacterial biofilms and multidrug-resistant bacteria. Several QSIs of natural origin have been identified, and in this study, several common food products and plants were extracted and screened for QSI activity in an attempt to isolate and characterize previously unknown QSI compounds active against the common opportunistic pathogen Pseudomonas aeruginosa. Several extracts displayed activity, but horseradish exhibited the highest activity. Chromatographic separation led to the isolation of a potent QSI compound that was identified by liquid chromatography-diode array detector-mass spectrometry (LC-DAD-MS) and nuclear magnetic resonance (NMR) spectroscopy as iberin-an isothiocyanate produced by many members of the Brassicaceae family. Real-time PCR (RT-PCR) and DNA microarray studies showed that iberin specifically blocks expression of QS-regulated genes in P. aeruginosa.     

Kin selection, quorum sensing and virulence in pathogenic bacteria

Bacterial growth and virulence often depends upon the cooperative release of extracellular factors excreted in response to quorum sensing (QS). We carried out an in vivo selection experiment in mice to examine how QS evolves in response to variation in relatedness (strain diversity), and the consequences for virulence. We started our experiment with two bacterial strains: a wild-type that both produces and responds to QS signal molecules, and a lasR (signal-blind) mutant that does not release extracellular factors in response to signal. We found that: (i) QS leads to greater growth within hosts; (ii) high relatedness favours the QS wild-type; and (iii) low relatedness favours the lasR mutant. Relatedness matters in our experiment because, at relatively low relatedness, the lasR mutant is able to exploit the extracellular factors produced by the cells that respond to QS, and hence increase in frequency. Furthermore, our results suggest that because a higher relatedness favours cooperative QS, and hence leads to higher growth, this will also lead to a higher virulence, giving a relationship between relatedness and virulence that is in the opposite direction to that usually predicted by virulence theory.     

The transcriptional regulator CzcR modulates antibiotic resistance and quorum sensing in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa responds to zinc, cadmium and cobalt by way of the CzcRS two-component system. In presence of these metals the regulatory protein CzcR induces the expression of the CzcCBA efflux pump, expelling and thereby inducing resistance to Zn, Cd and Co. Importantly, CzcR co-regulates carbapenem antibiotic resistance by repressing the expression of the OprD porin, the route of entry for these antibiotics. This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response. We found that CzcR affected the expression of numerous genes directly involved in the virulence of P. aeruginosa even in the absence of the inducible metals. Notably the full expression of quorum sensing 3-oxo-C12-HSL and C4-HSL autoinducer molecules is impaired in the absence of CzcR. In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay. Additionally, chromosome immunoprecipitation experiments allowed us to localize CzcR on the promoter of several regulated genes, suggesting a direct control of target genes such as oprD, phzA1 and lasI. All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.