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.     

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.     

Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2

Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence. This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers. While most autoinducers are species specific, autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi, is produced and detected by many Gram-negative and Gram-positive bacteria. The crystal structure of the V. harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester. Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium. We find that LsrB binds a chemically distinct form of the AI-2 signal, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), that lacks boron. Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling.     

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell–cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an “enhanced” strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 μM AI-2 to growing cell cultures, we determine that this “enhanced” AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.     

AI-2,一种新的细菌自体诱导分子

细菌通过数量阈值感应系统调节群体内个体的基因表达而使整个群体步调一致。细菌通过感应自体诱导分子(autoinducer,AI)浓度而感知周围环境中同类存在的密度,并据此调节自身特定性状的表达。AI．2是近年来新发现的一种介导细菌种间信号传导的自体诱导分子,其分子结构、功能均不同于传统的A1分子。AI．2对细菌的调节作用主要表现为对毒力基因表达的影响,但目前也有人认为AI-2可能只是一种代谢产物。     

Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2

Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.     

群体感应信号分子AI-2研究进展

群体感应(QS)是细菌根据种群密度的变化调控基因表达,协调群体行为的机制。除具有种特异性的信号分子AI-1外,近年来发现一类新的信号分子AI-2在调控细菌基因表达中起重要作用。AI-2的结构和生物合成途径已被确定,其产生依赖于一种称为LuxS的蛋白。目前认为AI-2在细菌种间交流中起通用信号分子(universalsignal)的作用。了解细菌的QS调控过程以及种间细胞交流的新机制,有助于对细菌病害进行防治。     

Chemotaxis to the quorum-sensing signal AI-2 requires the Tsr chemoreceptor and the periplasmic LsrB AI-2-binding protein

AI-2 is an autoinducer made by many bacteria. LsrB binds AI-2 in the periplasm, and Tsr is the l-serine chemoreceptor. We show that AI-2 strongly attracts Escherichia coli. Both LsrB and Tsr are necessary for sensing AI-2, but AI-2 uptake is not, suggesting that LsrB and Tsr interact directly in the periplasm.     

AI-2 biosynthesis module in a magnetic nanofactory alters bacterial response via localized synthesis and delivery

Nanofactories are nano-dimensioned and comprised of modules serving various functions that alter the response of targeted cells when deployed by locally synthesizing and delivering cargo to the surfaces of the targeted cells. In its basic form, a nanofactory consists of a minimum of two functional modules: a cell capture module and a synthesis module. In this work, magnetic nanofactories that alter the response of targeted bacteria by the localized synthesis and delivery of the "universal" bacterial quorum sensing signal molecule autoinducer AI-2 are demonstrated. The magnetic nanofactories consist of a cell capture module (chitosan-mag nanoparticles) and an AI-2 biosynthesis module that contains both AI-2 biosynthetic enzymes Pfs and LuxS on a fusion protein (His-LuxS-Pfs-Tyr, HLPT) assembled together. HLPT is hypothesized to be more efficient than its constituent enzymes (used separately) at conversion of the substrate SAH to product AI-2 on account of the proximity of the two enzymes within the fusion protein. HLPT is demonstrated to be more active than the constituent enzymes, Pfs and LuxS, over a wide range of experimental conditions. The magnetic nanofactories (containing bound HLPT) are also demonstrated to be more active than free, unbound HLPT. They are also shown to elicit an increased response in targeted Escherichia coli cells, due to the localized synthesis and delivery of AI-2, when compared to the response produced by the addition of AI-2 directly to the cells. Studies investigating the universality of AI-2 and unraveling AI-2 based quorum sensing in bacteria using magnetic nanofactories are envisioned. The prospects of using such multi-modular nanofactories in developing the next generation of antimicrobials based on intercepting and interrupting quorum sensing based signaling are discussed.     

Quorum sensing and swarming migration in bacteria

Bacterial cells can produce and sense signal molecules, allowing the whole population to initiate a concerted action once a critical concentration (corresponding to a particular population density) of the signal has been reached, a phenomenon known as quorum sensing. One of the possible quorum sensing-regulated phenotypes is swarming, a flagella-driven movement of differentiated swarmer cells (hyperflagellated, elongated, multinucleated) by which bacteria can spread as a biofilm over a surface. The glycolipid or lipopeptide biosurfactants thereby produced function as wetting agent by reducing the surface tension. Quorum sensing systems are almost always integrated into other regulatory circuits. This effectively expands the range of environmental signals that influence target gene expression beyond population density. In this review, we first discuss the regulation of AHL-mediated surface migration and the involvement of other low-molecular-mass signal molecules (such as the furanosyl borate diester AI-2) in biosurfactant production of different bacteria. In addition, population density-dependent regulation of swarmer cell differentiation is reviewed. Also, several examples of interspecies signalling are reported. Different signal molecules either produced by bacteria (such as other AHLs and diketopiperazines) or excreted by plants (such as furanones, plant signal mimics) might influence the quorum sensing-regulated swarming behaviour in bacteria different from the producer. On the other hand, specific bacteria can reduce the local available concentration of signal molecules produced by others. In the last part, the role and regulation of a surface-associated movement in biofilm formation is discussed. Here we also describe how quorum sensing may disperse existing biofilms and control the interaction between bacteria and higher organisms (such as the Rhizobium-bean symbiosis).     

A LuxP-based fluorescent sensor for bacterial autoinducer II.

Autoinducer 2 (AI-2), which enables different bacterial species to engage in interspecies communication, has been difficult to detect quantitatively. Currently, the most commonly used method for AI-2 detection employs an engineered Vibrio harveyi reporter strain, which produces bioluminescence in response to AI-2. However, the bioassay is not quantitative and is sensitive to assay conditions. In this work, we have developed two protein sensors for AI-2 by modifying AI-2 receptor proteins LuxP and LsrB with environmentally sensitive fluorescent dyes. The protein sensors bind specifically to AI-2 and produce dose-dependent changes in their fluorescence yield. The new assay method has been applied to monitor the enzymatic synthesis of AI-2 in real time and determine the extracellular and intracellular AI-2 concentrations in several bacterial culture fluids.     

The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram-negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram-negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI-1 and AI-2). AI-1 is a homoserine lactone. The structure of AI-2 is not known. We have suggested previously that V. harveyi uses AI-1 for intraspecies communication and AI-2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram-negative and Gram-positive bacteria produce AI-2 and, in every case, production of AI-2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI-2 synthase and that AI-2 is produced from S-adenosylmethionine in three enzymatic steps. The substrate for LuxS is S-ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI-2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI-2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI-2 is a unique, 'universal' signal that could be used by a variety of bacteria for communication among and between species.     

The LuxS-dependent autoinducer AI-2 controls the expression of an ABC transporter that functions in AI-2 uptake in Salmonella typhimurium

In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI-2, originally discovered in the quorum-sensing bacterium Vibrio harveyi, is made by many species of Gram-negative and Gram-positive bacteria. In every case, production of AI-2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI-2 in most of these luxS-containing species of bacteria are not known. Here, we describe the identification and characterization of AI-2-regulated genes in Salmonella typhimurium. We find that LuxS and AI-2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)-type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium. A gene encoding a DNA-binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI-2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI-2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI-2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI-2 into the cells. It is intriguing that an operon regulated by AI-2 encodes functions resembling the ribose transporter, given recent findings that AI-2 is derived from the ribosyl moiety of S-ribosylhomocysteine.     

Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios

Entry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogen Vibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNC V. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.     

Let LuxS speak up in AI-2 signaling

Quorum sensing is a process of bacterial cell-cell communication that uses small diffusible molecules to coordinate diverse behaviors in response to population density. The only quorum-sensing system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). The AI-2 synthase LuxS is widely distributed among the Bacteria, which suggests that AI-2 is a language for interspecies communication. However, LuxS is also an integral component of the activated methyl cycle in bacteria. LuxS-based quorum sensing has been intensively studied in the past decade, mostly in relation to the AI-2 molecule and the downstream effects of luxS knockouts; few studies have focused on the gene and protein activity itself. Ongoing attempts to dissect the metabolic and signaling roles of LuxS leave little doubt that unraveling the regulation of luxS expression and cellular LuxS activity is the key to understanding LuxS-based quorum sensing.     

Sinorhizobium meliloti, a bacterium lacking the autoinducer-2 (AI-2) synthase, responds to AI-2 supplied by other bacteria

Many bacterial species respond to the quorum-sensing signal autoinducer-2 (AI-2) by regulating different niche-specific genes. Here, we show that Sinorhizobium meliloti, a plant symbiont lacking the gene for the AI-2 synthase, while not capable of producing AI-2 can nonetheless respond to AI-2 produced by other species. We demonstrate that S. meliloti has a periplasmic binding protein that binds AI-2. The crystal structure of this protein (here named SmlsrB) with its ligand reveals that it binds (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), the identical AI-2 isomer recognized by LsrB of Salmonella typhimurium. The gene encoding SmlsrB is in an operon with orthologues of the lsr genes required for AI-2 internalization in enteric bacteria. Accordingly, S. meliloti internalizes exogenous AI-2, and mutants in this operon are defective in AI-2 internalization. S. meliloti does not gain a metabolic benefit from internalizing AI-2, suggesting that AI-2 functions as a signal in S. meliloti. Furthermore, S. meliloti can completely eliminate the AI-2 secreted by Erwinia carotovora, a plant pathogen shown to use AI-2 to regulate virulence. Our findings suggest that S. meliloti is capable of 'eavesdropping' on the AI-2 signalling of other species and interfering with AI-2-regulated behaviours such as virulence.     

Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli.

Recent reports have shown that bacterial cell-cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, the role of quorum sensing in nonpathogenic E. coli is less clear and, in particular, there is no information regarding the role of quorum sensing in overexpression of plasmid-encoded genes. In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E. coli. Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling. Importantly, our findings demonstrate for the first time that recombinant E. coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway.     

Identification of a key amino acid of LuxS involved in AI-2 production in Campylobacter jejuni

Autoinducer-2 (AI-2) mediated quorum sensing has been associated with the expression of virulence factors in a number of pathogenic organisms and has been demonstrated to play a role in motility and cytolethal distending toxin (cdt) production in Campylobacter jejuni. We have initiated the work to determine the molecular basis of AI-2 synthesis and the biological functions of quorum sensing in C. jejuni. In this work, two naturally occurring variants of C. jejuni 81116 were identified, one producing high-levels of AI-2 while the other is defective in AI-2 synthesis. Sequence analysis revealed a G92D mutation in the luxS gene of the defective variant. Complementation of the AI-2(-) variant with a plasmid encoded copy of the wild-type luxS gene or reversion of the G92D mutation by site-directed mutagenesis fully restored AI-2 production by the variant. These results indicate that the G92D mutation alone is responsible for the loss of AI-2 activity in C. jejuni. Kinetic analyses showed that the G92D LuxS has a ～100-fold reduced catalytic activity relative to the wild-type enzyme. Findings from this study identify a previously undescribed amino acid that is essential for AI-2 production by LuxS and provide a unique isogenic pair of naturally occurring variants for us to dissect the functions of AI-2 mediated quorum sensing in Campylobacter.