Inhibition of Pseudomonas aeruginosa quorum sensing by AI-2 analogs

Autoinducer-2 (AI-2) has been suggested to serve as a universal interspecies quorum sensing signaling molecule. We have synthesized a set of AI-2 analogs with small incremental changes in alkyl substitution on C-2 and evaluated them for their agonistic and antagonistic potential as quorum sensing (QS) attenuators in two different bacterial species: Pseudomonas aeruginosa and Vibrio harveyi. Unexpectedly, several of the analogs were found to function as synergistic QS agonists in V. harveyi, while two of these analogs inhibit QS in P. aeruginosa.     

Developing next generation antimicrobials by intercepting AI-2 mediated quorum sensing

Bacteria have been evolving antibiotic resistance since their discovery in the early twentieth century. Most new antibiotics are derivatives of older generations and there are now bacteria that are virtually resistant to almost all antibiotics. This poses a global threat to human health and has been classified as a clinical “super-challenge”, which has necessitated research into new antimicrobials that inhibit bacterial virulence while minimizing selective pressures that lead to the emergence of resistant strains. Quorum sensing (QS), the process of population dependent bacterial cell-cell signaling, can accelerate bacterial virulence and is an increasingly interesting target for developing next generation antimicrobials. Most QS inhibitors target species-specific signals, such as acylhomoserine lactones (AHLs) and oligopeptides. Methodologies for intercepting the cross-species signal, autoinducer-2 (AI-2), have only recently emerged. We review these strategies to prevent the relay of the AI-2 signal amongst pathogens, including Escherichia coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae and Pseudomonas aeruginosa. Inhibition mechanisms are categorized based on the target (i.e., enzymes for signal generation, the signal molecule itself, or the various components of the signal transduction process). The universal nature of the AI-2 signal imparts on its inhibitors the potential for broad spectrum use.     

Stereochemical diversity of AI-2 analogs modulates quorum sensing in Vibrio harveyi and Escherichia coli

Bacteria coordinate population-dependent behaviors such as virulence by intra- and inter-species communication (quorum sensing). Autoinducer-2 (AI-2) regulates inter-species quorum sensing. AI-2 derives from the spontaneous cyclisation of linear (S)-4,5-dihydroxypentanedione (DPD) into two isomeric forms in dynamic equilibrium. Different species of bacteria have different classes of AI-2 receptors (LsrB and LuxP) which bind to different cyclic forms. In the present work, DPD analogs with a new stereocenter at C-5 (4,5-dihydroxyhexanediones (DHDs)) have been synthesized and their biological activity tested in two bacteria. (4S,5R)-DHD is a synergistic agonist in Escherichia coli (which contains the LsrB receptor), while it is an agonist in Vibrio harveyi (LuxP), displaying the strongest agonistic activity reported so far (EC(50)=0.65μM) in this organism. Thus, modification at C-5 opens the way to novel methods to manipulate quorum sensing as a method for controlling bacteria.     

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.     

N-Acyl-homoserine lactones and autoinducer-2-mediated quorum sensing during wastewater treatment

Applied Microbiology and Biotechnology - Bacteria can coordinate and synchronize activities through a cell density-dependent regulatory mechanism called quorum sensing (QS). Bacteria can measure...     

嗜水气单胞菌群体感应信号分子AI-2的细胞外生物 合成及活性检测

【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对LuxS、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的LuxS分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏LuxS一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了LuxS、MtnN-1和MtnN-2蛋白,细胞外LuxS和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,LuxS和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与LuxS共同催化AI-2的细胞外生物合成。     

An efficient synthesis of the precursor of AI-2, the signalling molecule for inter-species quorum sensing

Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. A synthesis of (S)-4,5-dihydroxypentane-2,3-dione (DPD), the precursor of AI-2, is described starting from methyl glycolate. The key step was an asymmetric reduction of a ketone with (S)-Alpine borane. This new method was highly reproducible affording DPD for biological tests without contaminants. The biological activity was tested with the previously available assays and compared with a new method using an Escherichia coli reporter strain thus avoiding the use of the pathogenic Salmonella reporter.     

Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi

Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes were designed. Structural resemblances can be found between thiazolidinediones and well-known furanone type quorum sensing (QS) inhibitors such as N-acylaminofuranones, and/or acyl-homoserine lactone signaling molecules, while dioxazaborocanes structurally resemble previously reported oxazaborolidine derivatives which antagonized autoinducer 2 (AI-2) binding to its receptor. Because of this, we hypothesized that these compounds could affect AI-2 QS in Vibrio harveyi. Although all compounds blocked QS, the thiazolidinediones were the most active AI-2 QS inhibitors, with EC₅₀ values in the low micromolar range. Their mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of V. harveyi QS mutants and by DNA-binding assays with purified LuxR protein. The active compounds neither affected bioluminescence as such nor the production of AI-2. Instead, our results indicate that the thiazolidinediones blocked AI-2 QS in V. harveyi by decreasing the DNA-binding ability of LuxR. In addition, several dioxazaborocanes were found to block AI-2 QS by targeting LuxPQ.     

醋酸菌耐酸机理及其群体感应研究新进展

醋酸菌(acetic acid bacteria,AAB)是一类严格好氧的革兰氏阴性细菌,因其乙醇氧化生成醋酸能力强、高耐醋酸等特性而成为食醋发酵的主要工业菌种。醋酸菌的耐酸性对于高酸度食醋生产具有重要意义。随着醋酸菌的蛋白组学及基因组学研究的深入,其糖代谢、蛋白质代谢、脂代谢及应激响应等分子机制或过程也得到更多的阐释;葡糖醋杆菌中有关群体感应系统的研究报道则为从信号通路角度探索醋酸菌的耐酸机制提供了新的思路,进而对于高耐酸醋酸菌的选育以及醋酸发酵工艺的优化具重要的参考意义。本文在简介蛋白组、基因组研究的基础上,着重综述醋酸菌群体感应的研究进展。     

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。     

Evolution of commensal bacteria in the intestinal tract of mice

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甲烷古菌群感效应信号分子的检测

竹节状甲烷鬃菌(Methanosaeta harundinacea)6Ac是本实验室分离自厌氧颗粒污泥中的甲烷古菌新种。该菌具有短杆(3μm-5μm)和长链状(>200μm)两种细胞形态,且与细胞密度相关,暗示该菌可能存在群感效应调控的细胞形态变化。【目的】验证该菌存在群感效应信号分子并与细胞形态变化相关。【方法】用高丝氨酸内酯指示菌Agrobacterium tumefaciens NTL4检测菌株6Ac的培养液,并用购买的高丝氨酸内酯标准品加入短杆菌株6Ac检测形态变化。【结果】菌株6Ac的培养液中含有高丝氨酸内酯类物质。实验证明化学合成的高丝氨酸内酯N-(β-酮基)辛酰高丝氨酸内酯能够促进竹节状甲烷鬃菌的长链细胞形成。而且在马氏甲烷八叠球菌(Methanosarcina mazei)、热自养甲烷杆菌(Methanothermobacter thermautotrophicus)和甲酸甲烷杆菌(Methanobacterium formicicum)的培养液中也检测到了高丝氨酸内酯。【结论】多种甲烷古菌可以产生高丝氨酸内酯类物质,并可能以此类物质作为群感效应的信号分子。     

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.     

Stimulating cROSstalk between commensal bacteria and intestinal stem cells

EMBO J (2013) 32 23, 3017–3028 10.1038/emboj.2013.224; published online October182013Commensal gut bacteria benefit their host in many ways, for instance by aiding digestion and producing vitamins. In a new study in The EMBO Journal, Jones et al (2013) report that commensal bacteria can also promote intestinal epithelial renewal in both flies and mice. Interestingly, among commensals this effect is most specific to Lactobacilli, the friendly bacteria we use to produce cheese and yogurt. Lactobacilli stimulate NADPH oxidase (dNox/Nox1)-dependent ROS production by intestinal enterocytes and thereby activate intestinal stem cells.The human gut contains huge numbers of bacteria (∼10¹⁴/person) that play beneficial roles for our health, including digestion, building our immune system and competing with harmful microbes (Sommer and Backhed, 2013). Both commensal and pathogenic bacteria can elicit antimicrobial responses in the intestinal epithelium and also stimulate epithelial turnover (Buchon et al, 2013; Sommer and Backhed, 2013). In contrast to gut pathogens, relatively little is known about how commensal bacteria influence intestinal turnover. In a simple yet elegant study reported recently in The EMBO Journal, Jones et al (2013) show that among several different commensal bacteria tested, only Lactobacilli promoted much intestinal stem cell (ISC) proliferation, and it did so by stimulating reactive oxygen species (ROS) production. Interestingly, the specific effect of Lactobacilli was similar in both Drosophila and mice. In addition to distinguishing functional differences between species of commensals, this work suggests how the ingestion of Lactobacillus-containing probiotic supplements or food (e.g., yogurt) might support epithelial turnover and health.In both mammals and insects, ISCs give rise to intestinal enterocytes, which not only absorb nutrients from the diet but must also interact with the gut microbiota (Jiang and Edgar, 2012). The metazoan intestinal epithelium has developed conserved responses to enteric bacteria, for instance the expression of antimicrobial peptides (AMPs; Gallo and Hooper, 2012; Buchon et al, 2013), presumably to kill harmful bacteria while allowing symbiotic commensals to flourish. In addition to AMPs, intestinal epithelial cells use NADPH family oxidases to generate ROS that are used as microbicides (Lambeth and Neish, 2013). High ROS levels during enteric infections likely act non-discriminately against both commensals and pathogens, but controlled, low-level ROS can act as signalling molecules that regulate various cellular processes including proliferation (Lambeth and Neish, 2013). In flies, exposure to pathogenic Gram-negative bacteria has been reported to result in ROS (H₂O₂) production by an enzyme called dual oxidase (Duox; Ha et al, 2005). Duox activity in the fly intestine (and likely also the mammalian one) has recently been discovered to be stimulated by uracil secretion by pathogenic bacteria (Lee et al, 2013). In the mammalian intestine another enzyme, NADPH oxidase (Nox), has also been shown to produce ROS in the form of superoxide (O₂⁻), in this case in response to formylated bacterial peptides (Lambeth and Neish, 2013). A conserved role for Nox in the Drosophila intestinal epithelium had not until now been explored.Jones et al (2013) checked seven different commensal bacterial to see which would stimulate ROS production by the fly''s intestinal epithelium, and found that only one species, a Gram-positive Lactobacillus, could stimulate significant production of ROS in intestinal enterocytes. Five bacterial species were checked in mice or cultured intestinal cells, and again it was a Lactobacillus that generated the strongest ROS response. Although not all of the most prevalent enteric bacteria were assayed, those others that were—such as E. coli—induced only mild, barely detectable levels of ROS in enterocytes. Surprisingly, although bacteria pathogenic to Drosophila, like Erwinia caratovora, were expected to stimulate ROS production via Duox, Jones et al (2013) did not observe this using the ROS detecting dye hydrocyanine-Cy3, or a ROS-sensitive transgene reporter, Glutatione S-transferase-GFP, in flies. Further, Jones et al (2013) found that genetically suppressing Nox in either Drosophila or mice decreased ROS production after Lactobacillus ingestion. Consistent with the important role of Nox, Duox appeared not to be required for ROS production after Lactobacillus ingestion. In addition, Jones et al (2013) found that Lactobacilli also promoted DNA replication—a metric of cell proliferation and epithelial renewal—in the fly''s intestine, and that this was also ROS- and Nox-dependent. Again, the same relationship was found in the mouse small intestine. Together, these results suggest a conserved mechanism by which Lactobacilli can stimulate Nox-dependent ROS production in intestinal enterocytes and thereby promote ISC proliferation and enhance gut epithelial renewal.In the fly midgut, uracil produced by pathogenic bacteria can stimulate Duox-dependent ROS production, which is thought to act as a microbicide (Lee et al, 2013), and can also promote ISC proliferation (Buchon et al, 2009). However, Duox-produced ROS may also damage the intestinal epithelium itself and thereby promote epithelial regeneration indirectly through stress responses. In this disease scenario, ROS appears to be sensed by the stress-activated Jun N-terminal Kinase (JNK; Figure 1A), which can induce pro-proliferative cytokines of the Leptin/IL-6 family (Unpaireds, Upd1–3) (Buchon et al, 2009; Jiang et al, 2009). These cytokines activate JAK/STAT signalling in the ISCs, promoting their growth and proliferation, and accelerating regenerative repair of the gut epithelium (Buchon et al, 2009; Jiang et al, 2009). It is also possible, however, that low-level ROS, or specific types of ROS (e.g., H₂O₂) might induce ISC proliferation directly by acting as a signal between enterocytes and ISCs. Since commensal Lactobacillus stimulates ROS production via Nox rather than Duox, this might be a case in which a non-damaging ROS signal promotes intestinal epithelial renewal without stress signalling or a microbicidal effect (Figure 1B). However, Jones et al (2013) stopped short of ruling out a role for oxidative damage, cell death or stress signalling in the intestinal epithelium following colonization by Lactobacilli, and so these parameters must be checked in future studies. Perhaps even the friendliest symbiotes cause a bit of ‘healthy'' damage to the gut lining, stimulating it to refresh and renew. Whether damage-dependent or not, the stimulation of Drosophila ISC proliferation by commensals and pathogens alike appears to involve the same cytokine (Upd3; Buchon et al, 2009), and so some of the differences between truly pathogenic and ‘friendly'' gut microbes might be ascribed more to matters of degree than qualitative distinctions. Future studies exploring exactly how different types of ROS signals stimulate JNK activity, gut cytokine expression and epithelial renewal should be able to sort this out, and perhaps help us learn how to better manage the ecosystems in our own bellies. From the lovely examples reported by Jones et al (2013), an experimental back-and-forth between the Drosophila and mouse intestine seems an informative way to go.Open in a separate windowFigure 1Metazoan intestinal epithelial responses to commensal and pathogenic bacteria. (A) High reactive oxygen species (ROS) levels generated by dual oxidase (Duox) in response to uracil secretion by pathogenic bacteria. (B) Low ROS levels generated by NADPH oxidase (Nox) in response to commensal bacteria. In addition to acting as a microbiocide, ROS in flies may stimulate JNK signaling and cytokine (Upd 1–3) expression in enterocytes, thereby stimulating ISC proliferation and epithelial turnover or regeneration. Whether this stimulation required damage to or loss of enterocytes has yet to be explored.     

黄河鲤水产细菌的分离及其毒力因子和群体感应信号分子的检测

[背景]水产细菌病害制约水产养殖业健康发展,群体感应与细菌毒力因子的产生密切相关,群体感应调控细菌的毒力因子特性值得进一步研究.[目的]探究群体感应与黄河鲤细菌病害的关系,明确群体感应对细菌毒力因子特性的影响.[方法]通过16SrRNA基因测序并构建系统进化树确定筛选菌株的进化地位,通过脱脂牛奶平板法和偶氮酪蛋白法检测...     

AI-2 does not function as a quorum sensing molecule inCampylobacter jejuniduring exponential growthin vitro

Background  

Campylobacter jejunicontains a homologue of theluxSgene shown to be responsible for the production of the signalling molecule autoinducer-2 (AI-2) inVibrio harveyiandVibrio cholerae. The aim of this study was to determine whether AI-2 acted as a diffusible quorum sensing signal controllingC. jejunigene expression when it is produced at high levels during mid exponential growth phase.     

Detection of quorum sensing signal molecules in the family Vibrionaceae

Aims: The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N‐acyl‐homoserine lactone (AHL), the autoinducer‐2 (AI‐2) and the cholerae autoinducer‐1‐like (CAI‐1‐like) molecules in 25 Vibrionaceae strains. Methods and Results: The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty‐one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin‐layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI‐2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI‐1‐like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. Conclusions: The results indicated that the QS signal molecules, especially AHL and AI‐2 molecules, were widespread in the family Vibrionaceae. Significance and Impact of the Study: In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.     

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.