Bacterial interspecies quorum sensing in the mammalian gut microbiota

The mammalian gastrointestinal tract harbors a diverse and complex resident bacterial community, which interacts with the host in many beneficial processes required for optimal host health. We are studying the importance of bacterial cell-cell communication mediated by the interspecies quorum-sensing signal autoinducer-2 (AI-2) in the beneficial properties of the gut microbiota. Our recent work provided the first evidence that AI-2 produced by Escherichia coli can influence the species composition of this community in the mouse gut. We showed that, under conditions of microbiota imbalances induced by antibiotic treatments, E. coli, which increases intestinal AI-2 levels, not only had an effect on the overall structure of the microbiota community, but specifically favored the expansion of the Firmicutes phylum. Because the Firmicutes are very important for many gut functions and were the group of bacteria most severely affected by antibiotic treatment with streptomycin, we are addressing the possibility that AI-2 can influence the balance of the major bacterial groups in the gut and promote recovery of gut homeostasis. Overall, we want to understand how bacterial chemical signaling shapes the multi-species bacterial communities in the mammalian gut and how these communities affect host physiology.     

Synthesis of d-desthiobiotin-AI-2 as a novel chemical probe for autoinducer-2 quorum sensing receptors

In processes regulated by quorum sensing (QS) bacteria respond to the concentration of autoinducers in the environment to engage in group behaviours. Autoinducer-2 (AI-2) is unique as it can foster interspecies communication. Currently, two AI-2 receptors are known, LuxP and LsrB, but bacteria lacking these receptors can also respond to AI-2. In this work, we present an efficient and reproducible synthesis of a novel chemical probe, d-desthiobiotin-AI-2. This probe binds both LuxP and LsrB receptors from different species of bacteria. Thus, this probe is able to bind receptors that recognise the two known biologically active forms of AI-2, presenting the plasticity essential for the identification of novel unknown AI-2 receptors. Moreover, a protocol to pull down receptors bound to d-desthiobiotin-AI-2 with anti-biotin antibodies has also been established. Altogether, this work highlights the potential of conjugating chemical signals to biotinylated derivatives to identify and tag signal receptors involved in quorum sensing or other chemical signalling processes.     

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.     

Dihydropyrrolones as bacterial quorum sensing inhibitors

Bacteria regulate their pathogenicity and biofilm formation through quorum sensing (QS), which is an intercellular communication system mediated by the binding of signaling molecules to QS receptors such as LasR. In this study, a range of dihydropyrrolone (DHP) analogues were synthesized via the lactone-lactam conversion of lactone intermediates. The synthesized compounds were tested for their ability to inhibit QS, biofilm formation and bacterial growth of Pseudomonas aeruginosa. The compounds were also docked into a LasR crystal structure to rationalize the observed structure-activity relationships. The most active compound identified in this study was compound 9i, which showed 63.1% QS inhibition of at 31.25?µM and 60% biofilm reduction at 250?µM with only moderate toxicity towards bacterial cell growth.     

A new phenothiazine structural scaffold as inhibitors of bacterial quorum sensing in Vibrio harveyi

Quorum sensing has attracted much attention due to its involvement in pathologically relevant events such as biofilm formation, virulence factor production, and sporulation. Inhibitors of quorum sensing are important research tools and potential therapeutic agents. In this paper, we describe a phenothiazine structural scaffold as a new type of quorum sensing inhibitors with IC₅₀ values in the single digit micro molar range in Vibrio harveyi.     

A sensitive fluorescence reporter for monitoring quorum sensing regulated protease production in Vibrio harveyi

Many bacteria produce and secrete proteases during host invasion and pathogenesis. Vibrio harveyi, an opportunistic pathogen of shrimp, is known to use a two-component quorum sensing (QS) mechanism for coordination of gene expression including protease secretion at high population densities. We examined the role of V. harveyi's QS signaling molecules, N-(3-hydroxybutanoyl)-l-homoserine lactone (AI-1) and the boron derivative of autoinducer-2 (BAI-2) in extracellular protease production. A fusion protein, M3CLPY (Rajamani et al., 2007), consisting of a large protease sensitive BAI-2 mutant receptor LuxP (~ 38 kDa) flanked by two protease insensitive cyan and yellow variants of GFP (~ 28 kDa each) was utilized as a substrate to detect secreted protease activity. The M3CLPY fusion, with the addition of wild-type V. harveyi (BB120) cell-free culture filtrate showed a time-dependent loss in fluorescence resonance energy transfer (FRET) associated with the cleavage of the LuxP linker protein and hence separation of the two flurophores. This cleavage of LuxP linker protein leading to decreased FRET efficiency was further confirmed by immunoblotting using anti-GFP antibody. The addition of cell-free filtrates from strains defective in one or both of the two-component QS pathways: luxN⁻ (defective in AI-1), luxS⁻ (defective in BAI-2), and luxN⁻/luxS⁻ (defective in both AI-1/BAI-2) showed differential levels of protease production. The observed protease activities were most pronounced in wild-type, followed by the AI-1 defective mutant (BB170) and the least for luxS⁻ mutant (MM30) and luxN⁻/luxS⁻ double mutant (MM32) strains. Incidentally, the lowest protease producing strains MM30 and MM32 were both defective in BAI-2 production. This observation was validated by addition of synthetic BAI-2 to MM30 and MM32 strains to restore protease production. Our results indicate that BAI-2 signaling in the two-component QS pathway plays the key role in regulating extracellular protease production in V. harveyi.     

铜绿假单胞菌群体感应抑制剂研究进展

铜绿假单胞菌是一种常见的机会致病菌,可在人群中引起严重的急性和慢性感染,是病人在医院期间发生感染的第三大致病菌。铜绿假单胞菌多种毒力因子的分泌以及生物被膜的形成都是受一种被称为群体感应(Quorum Sensing,QS)的胞间信号传导系统调控的。QS使细菌能够根据细胞密度变化进行基因表达的调控。通过抑制QS来治疗铜绿假单胞菌感染是一个很有前景的发展方向。本文将就近年来铜绿假单胞菌群体感应抑制剂的研究进展进行综述。     

Computational modeling of differences in the quorum sensing induced luminescence phenotypes of Vibrio harveyi and Vibrio cholerae

Vibrio harveyi and Vibrio cholerae have quorum sensing pathways with similar design and highly homologous components including multiple small RNAs (sRNAs). However, the associated luminescence phenotypes of strains with sRNA deletions differ dramatically: in V. harveyi, the sRNAs act additively; however, in V. cholerae, the sRNAs act redundantly. Furthermore, there are striking differences in the luminescence phenotypes for different pathway mutants in V. harveyi and V. cholerae. However, these differences have not been connected with the observed differences for the sRNA deletion strains in these bacteria. In this work, we present a model for quorum sensing induced luminescence phenotypes focusing on the interactions of multiple sRNAs with target mRNA. Within our model, we find that one key parameter - the fold-change in protein concentration necessary for luminescence activation - can control whether the sRNAs appear to act additively or redundantly. For specific parameter choices, we find that differences in this key parameter can also explain hitherto unconnected luminescence phenotypes differences for various pathway mutants in V. harveyi and V. cholerae. The model can thus provide a unifying explanation for observed differences in luminescence phenotypes and can also be used to make testable predictions for future experiments.     

Synthesis and biological activity of a potent optically pure autoinducer-2 quorum sensing agonist

Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.     

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。     

密度感应系统对弗氏志贺菌生长竞争能力的影响

密度感应系统调节细菌应答反应的发生,这些应答反应与细胞密度有关。通过对比大肠杆菌(Escherichia coli)和志贺氏菌(Shigella spp.)的序列发现,志贺菌属密度感应系统操纵子普遍存在丢失或突变。为研究其密度感应系统的功能,文章利用哈氏弧菌(Vibrio harveyi)BB170作为指示菌,检测弗氏志贺菌(Shigella flexneri)密度感应系统信号分子AI-2,证明其可以分泌有活性的AI-2;其次,采用Golden Gate克隆法将大肠杆菌MG1655的密度感应系统基因克隆至弗氏志贺菌301中,获得密度感应系统回复株301。通过菌落计数表明,在混合培养条件下,密度感应系统基因回复株301比野生株301存在生长优势;通过双向电泳初步比较分析表明,密度感应系统基因可以在志贺菌中表达,并鉴定到了其他一些与应激反应相关的差异表达蛋白, 如Hsp60、GroEL、SodB。     

Boronic acids as inhibitors of steroid sulfatase

Steroid sulfatase (STS) catalyzes the hydrolysis of steroidal sulfates such as estrone sulfate (ES1) to the corresponding steroids and inorganic sulfate. STS is considered to be a potential target for the development of therapeutics for the treatment of steroid-dependent cancers. Two steroidal and two coumarin- and chromenone-based boronic acids were synthesized and examined as inhibitors of purified STS. The boronic acid analog of estrone sulfate bearing a boronic acid moiety at the 3-position in place of the sulfate group was a good competitive STS inhibitor with a K_i of 2.8 μM at pH 7.0 and 6.8 μM at pH 8.8. The inhibition was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. An estradiol derivative bearing a boronic acid group at the 3-position and a benzyl group at the 17-position was a potent reversible, non-competitive STS inhibitor with a K_i of 250 nM. However, its 3-OH analog, a known STS inhibitor, exhibited an almost identical affinity for STS and also bound in a non-competitive manner. It is suggested that these compounds prefer to bind in a hydrophobic tunnel close to the entrance to the active site. The coumarin and chromenone boronic acids were modest inhibitors of STS with IC₅₀s of 86 and 171 μM, respectively. Surprisingly, replacing the boronic acid group of the chromenone derivative with an OH group yielded a good reversible, mixed type inhibitor with a K_i of 4.6 μM. Overall, these results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog.     

群体感应抑制剂对海洋生态功能菌生物膜形成的影响

[目的]研究天然群体感应抑制剂(Quorum sensing inhibitors,QSI)分子对海洋生态功能菌生物膜形成的影响.[方法]以对污损生物幼虫附着具有诱导作用的海洋细菌为目标菌,通过在其生物膜的形成过程中添加天然群体感应抑制剂,研究其对目标菌成膜细菌数和浮游细菌数、生物膜形态以及生物膜表面胞外多糖含量的影响.[结果]呋喃酮和吡啶在50 mg/L时,对8株目标菌的成膜有显著的抑制作用,抑制率在80％左右,吲哚、青霉烷酸和香豆素在较高浓度800 mg/L才有比较好的抑制活性.生长抑制实验结果显示,同等浓度下,QSI分子对目标菌成膜的抑制活性明显高于其对浮游细菌生长的抑制活性.结果表明,QSI分子主要通过干扰目标菌群体感应系统以抑制生物膜的形成.[结论]研究证实QSI分子在海洋菌生物膜形成过程中具有一定的调控作用.通过添加QSI可能能够间接抑制由生物膜诱导的污损生物附着,从而以新的角度研制新型抗污损物质.     

Pyrogallol and its analogs can antagonize bacterial quorum sensing in Vibrio harveyi

Bacteria can coordinate community-wide behaviors through quorum sensing, that is, the secretion and sensing of autoinducer (AI) molecules. Bacterial quorum sensing is implicated in the regulation of pathologically relevant events such as biofilm formation, bacterial virulence, and drug resistance. Inhibitors of bacterial quorum sensing could therefore be useful therapeutics. Herein we report for the first time the discovery of several pyrogallol compounds as single digit micromolar inhibitors of bacterial quorum sensing in Vibrio harveyi.     

Synthetic nanoparticles for selective hydrolysis of bacterial autoinducers in quorum sensing

N-acyl homoserine lactones (AHLs) are signal molecules used by a large number of gram-negative bacteria in quorum sensing and their hydrolysis is known to inhibit biofilm formation. Micellar imprinting of AHL-like templates with catalytic functional monomers yielded water-soluble nanoparticles with AHL-shaped active site and nearby catalytic groups. Either Lewis acidic zinc ions or nucleophilic pyridyl ligands could be introduced through this strategy, yielding artificial enzymes for the hydrolysis of AHLs in a substrate-selective fashion.     

Interplay of two quorum sensing regulation systems of Vibrio fischeri

Many bacteria developed a possibility to recognise aspects of their environment or to communicate with each other by chemical signals. An important strategy is the so-called quorum sensing (QS), a regulatory mechanism for the gene expression, where the bacteria measure their own cell density by means of this signalling pathway. One of the best-studied species using QS is the marine luminescent bacterium Vibrio fischeri which is considered here as a model organism.The two main regulatory pathways (lux and ain) are combined to a regulation system, the dynamics is modelled by an ODE system. This system is analysed thoroughly, considering stationary states, dynamical behaviour and the possible biological meaning of it. The influence of different parameter values on the behaviour is examined, the same basic system is able to reflect the peculiarities of different bacteria strains (respectively, their mutants).     

The role of quorum sensing and the effect of environmental conditions on biofilm formation by strains of Vibrio vulnificus

Abstract

It has been suggested that Vibrio vulnificus attaches to plankton and algae and is found in large numbers in the environment. Factors affecting attachment, biofilm formation and morphology of V. vulnificus have not been thoroughly investigated. This study evaluated the role of quorum sensing (QS) and environmental conditions on biofilm development of V. vulnificus. It was found that biofilm development by V. vulnificus was affected by nutrient and glucose concentration, but not by NaCl concentration or temperature under the conditions used here. Moreover, biofilm development of a QS mutant strain proceeded rapidly and sloughing occurred earlier than for the isogenic parent strain. There was a significant loss of viability for the QS mutant biofilm early in development. Hence, it is hypothesised that factors regulated by the QS system play a role in proper biofilm development and maintenance of V. vulnificus. Furthermore, it is shown that biofilm development varied among isolates.     

Inactivation of bacterial quorum sensing signals N-acyl homoserine lactones is widespread in yeasts

The inactivation of quorum sensing signals, a phenomenon known as quorum quenching, has been described in diverse microorganisms, though it remains almost unexplored in yeasts. Beyond the well-known properties of these microorganisms for the industry or as eukaryotic models, the role of yeasts in soil or in the inner tissues of a plant is largely unknown. In this report, the wider survey of quorum quenching activities in yeasts isolated from Antarctic soil and the inner tissues of sugarcane, a tropical crop, is presented. Results show that, independently of their niche, quorum quenching activities are broadly present in unicellular fungi. Although yeasts showing a broad range of quorum quenching activity are present in the two niches, at the same time specific AHL inactivation profiles can also be found. Furthermore, yeasts from both sampling sites show quorum quenching activities compatible with lactonase-like and acylase-like inactivations of AHLs. Interestingly, the characterization of Rhodotorula mucilaginosa 7Apo1 showed that the presence of a particular AHL does not interfere with the quenching of a second molecule. Evidence suggests that yeasts could play a role in the modulation of the quorum sensing activity of bacteria. The relationship among phylogeny, sampling sites and yeast quorum quenching activities of the isolates is analyzed.     

Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning

Quorum sensing (QS) controls the pathogenic behavior of Streptococcus mutans, a primary cause of dental caries. S. mutans uses the competence stimulating peptide (CSP) to control mutacin production, a bacteriocin utilized by S. mutans to outcompete different commensal bacteria in mixed biofilm environments. In this study, we performed an N-methyl scan of an 18-CSP-based scaffold lacking the first two amino acid residues that were shown to be dispensable, to gain important mechanistic insight as to the role of backbone amide protons in the interaction between CSP and the ComD receptor. We then utilized the reverse alanine approach to develop CSP-based analogs with enhanced activities. The two most potent analogs were found to induce bacteriocin production at sub-nanomolar concentration using an interspecies inhibition assay. Overall, our analysis revealed that the 18-CSP sequence is not optimized and can be improved by replacement of multiple positions with alanine. Our results further suggest that the hydrophobic residues in S. mutans 18-CSP are involved in both receptor binding and activation.     

具有群体感应抑制活性的海洋细菌的筛选

【目的】从海洋环境中筛选出能有效抑制细菌群体感应的活性菌株,为以致病菌群体感应为靶点的新型疗法提供新的天然产物资源。【方法】以紫色杆菌(Chromobacteriumviolaceum)为报告菌,采用滤纸片法和双层软琼脂法相结合的筛选模型进行群体感应抑制活性菌的筛选。【结果】通过对美国圣璜岛海域海绵中分离出来的272株海洋细菌群体感应抑制活性的筛选,得到了具有抑制紫色杆菌素产生的细菌51株,其中74号菌株抑制效果最好,具有进一步研究的价值。【结论】海洋细菌中有很多具有抑制细菌群体感应效应的菌株,是天然群体感应抑制剂的潜在来源。