Lian-Hui Wang

Quenching quorum-sensing-dependent bacterial infection by an N -acyl homoserine lactonase

Bacterial cells sense their population density through a sophisticated cell–cell communication system and trigger expression of particular genes when the density reaches a threshold. This type of gene regulation, which controls diverse biological functions including virulence, is known as quorum sensing. Quorum-sensing signals, such as acyl-homoserine lactones (AHLs), are the essential components of the communication system. AHLs regulate virulence gene expression in a range of plant and animal (including human) bacterial pathogens. AHL-producing tobacco restored the pathogenicity of an AHL-negative mutant of Erwinia carotovora. Different bacterial species may produce different AHLs, which vary in the length and substitution of the acyl chain but contain the same homoserine lactone moiety. Here we show that the acyl-homoserine lactonase (AHL-lactonase), a new enzyme from Bacillus sp., inactivates AHL activity by hydrolysing the lactone bond of AHLs. Plants expressing AHL-lactonase quenched pathogen quorum-sensing signalling and showed significantly enhanced resistance to E. carotovora infection. Our results highlight a promising potential to use quorum-sensing signals as molecular targets for disease control, thereby broadening current approaches for prevention of bacterial infections.

Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes

N ‐acylhomoserine lactones (AHLs) are used as signal molecules by many quorum‐sensing Proteobacteria. Diverse plant and animal pathogens use AHLs to regulate infection and virulence functions. These signals are subject to biological inactivation by AHL‐lactonases and AHL‐acylases. Previously, little was known about the molecular details underlying the latter mechanism. An AHL signal‐inactivating bacterium, identified as a Ralstonia sp., was isolated from a mixed‐species biofilm. The signal inactivation encoding gene from this organism, which we call aiiD , was cloned and successfully expressed in Escherichia coli and inactivated three AHLs tested. The predicted 794‐amino‐acid polypeptide was most similar to the aculeacin A acylase (AAC) from Actinoplanes utahensis and also shared significant similarities with cephalosporin acylases and other N‐terminal (Ntn) hydrolases. However, the most similar homologues of AiiD are deduced proteins of undemonstrated function from available Ralstonia , Deinococcus and Pseudomonas genomes. LC‐MS analyses demonstrated that AiiD hydrolyses the AHL amide, releasing homoserine lactone and the corresponding fatty acid. Expression of AiiD in Pseudomonas aeruginosa PAO1 quenched quorum sensing by this bacterium, decreasing its ability to swarm, produce elastase and pyocyanin and to paralyse nematodes. Thus, AHL‐acylases have fundamental implications and hold biotechnological promise in quenching quorum sensing.

A bacterial cell–cell communication signal with cross-kingdom structural analogues

Extracellular signals are the key components of microbial cell–cell communication systems. This report identified a diffusible signal factor (DSF), which regulates virulence in Xanthomonas campestris pv. campestris, as cis‐11‐methyl‐2‐dodecenoic acid, an α,β unsaturated fatty acid. Analysis of DSF derivatives established the double bond at the α,β positions as the most important structural feature for DSF biological activity. A range of bacterial pathogens, including several Mycobacterium species, also displayed DSF‐like activity. Furthermore, DSF is structurally and functionally related to farnesoic acid (FA), which regulates morphological transition and virulence by Candida albicans, a fungal pathogen. Similar to FA, which is also an α,β unsaturated fatty acid, DSF inhibits the dimorphic transition of C. albicans at a physiologically relevant concentration. We conclude that α,β unsaturated fatty acids represent a new class of extracellular signals for bacterial and fungal cell–cell communications. As prokaryote–eukaryote interactions are ubiquitous, such cross‐kingdom conservation in cell–cell communication systems might have significant ecological and economic importance.

Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens

A signal turnover system is an essential component of many genetic regulatory mechanisms. The best-known example is the ubiquitin-dependent protein degradation system that exists in many organisms. We found that Agrobacterium tumefaciens adopts a unique signal turnover system to control exiting from a quorum-sensing mode. A. tumefaciens regulates Ti plasmid conjugal transfer by a quorum-sensing signal, N-3-oxo-octanoyl homoserine lactone (3OC8HSL), also known as Agrobacterium autoinducer. By using Tn5 mutagenesis and a functional cloning approach, we identified two genes that are involved in switching from a conjugal quorum-sensing mode to a nonconjugal mode at the onset of stationary phase. First, we located attJ, which codes for an IclR-type suppressor that regulates the second gene attM. The latter encodes a homologue of N-acylhomoserine lactone (AHL)-lactonase. Mass spectrometry analysis shows that the enzyme encoded by attM is an AHL-lactonase that hydrolyzes the lactone ring of 3OC8HSL. In wild-type A. tumefaciens, attM expression is initially suppressed by AttJ but significantly elevated at the stationary phase accompanied a sharp decline in 3OC8HSL. DNA gel retardation analysis shows that AttJ specifically binds to the promoter that controls AHL-lactonase expression. Mutation of attJ resulted in constitutive production of AHL-lactonase that abolishes 3OC8HSL accumulation and Ti plasmid transfer. These data suggest that A. tumefaciens has a sophisticated multicomponent quorum-sensing signal turnover system, allowing the cell to sense a change in growth and adjust cellular activities accordingly.

Quorum-quenching microbial infections: mechanisms and implications

The discovery of antibiotics early in the past century marked the beginning of active control and prevention of infectious microbial diseases. However, extensive use of antibiotics has also unavoidably resulted in the emergence of ‘superbugs’ that resist conventional antibiotics. The finding that many pathogens rely on cell-to-cell communication mechanisms, known as quorum sensing, to synchronize microbial activities essential for infection and survival in the host suggests a promising disease control strategy, i.e. quenching microbial quorum sensing or in short, quorum quenching. Work over the past few years has demonstrated that quorum-quenching mechanisms are widely conserved in many prokaryotic and eukaryotic organisms. These naturally occurring quorum-quenching mechanisms appear to play important roles in microbe–microbe and pathogen–host interactions and have been used, or served as lead compounds, in developing and formulating a new generation of antimicrobials. Characterization of the crystal structures of several types of quorum-quenching enzymes has provided valuable information to elucidate the catalytic mechanisms, as well as clues for future protein tailoring and molecular improvement. The discovery of quorum-sensing signal degradation enzymes in mammalian species represents a new milestone in quorum sensing and quorum quenching research. The finding highlights the importance of investigating their roles in host innate defence against infectious diseases and to determine the factors influencing their in vivo concentrations and catalytic activities.

A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition

In addition to producing lethal antibiotics, microorganisms may also use a new form of antagonistic mechanism in which signal molecules are exported to influence the gene expression and hence the ecological competence of their competitors. We report here the isolation and characterization of a novel signaling molecule, cis-2-dodecenoic acid (BDSF), from Burkholderia cenocepacia. BDSF is structurally similar to the diffusible signal factor (DSF) that is produced by the RpfF enzyme of Xanthomonas campestris. Deletion analysis demonstrated that Bcam0581, which encodes an RpfF homologue, was essential for BDSF production. The gene is highly conserved and widespread in the Burkholderia cepacia complex. Exogenous addition of BDSF restored the biofilm and extracellular polysaccharide production phenotypes of Xanthomonas campestris pv. campestris DSF-deficient mutants, highlighting its potential role in inter-species signaling. Further analyses showed that Candida albicans germ tube formation was strongly inhibited by either coculture with B. cenocepacia or by exogenous addition of physiological relevant levels of BDSF, whereas deletion of Bcam0581 abrogated the inhibitory ability of the bacterial pathogen. As B. cenocepacia and C. albicans are frequently encountered human pathogens, identification of the BDSF signal and its activity thus provides a new insight into the molecular grounds of their antagonistic interactions whose importance to microbial ecology and pathogenesis is now becoming evident.

Quorum quenching enzyme activity is widely conserved in the sera of mammalian species

Acyl‐homoserine lactone (AHL) quorum sensing signals play a key role in synchronizing virulence gene expression in Pseudomonas aeruginosa, which could cause fatal bloodstream infections. We showed that AHL inactivation activity, albeit with variable efficiency, was conserved in the serum samples of all the 6 tested mammalian animals. High‐performance liquid chromatography and mass spectrometry analyses revealed that mammalian sera had a lactonase‐like enzyme(s), which hydrolyzed the lactone ring of AHL to produce acyl homoserine, with enzyme properties reminiscent of paraoxonases (PONs). We further showed that the animal cell lines expressing three mouse PON genes, respectively, displayed strong AHL degradation activities.

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions.

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis‐11‐methyl‐2‐dodecenoic acid, as a cell–cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF‐mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild‐type strain XC1 and its DSF‐deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF‐dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell–cell signalling is not only essential for co‐ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.

Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network.

The bacterial pathogen Xanthomonas campestris pv. campestris co‐ordinates virulence factor production and biofilm dispersal through a diffusible signal factor (DSF)‐mediated cell–cell communication mechanism. The RpfC/RpfG two‐component system plays a key role in DSF signal transduction and appears to modulate downstream DSF regulon by changing intracellular content of cyclic dimeric GMP (c‐di‐GMP), an unusual nucleotide second messenger. Here we show that Clp, a conserved global regulator showing a strong homology to the cAMP nucleotide receptor protein Crp of Escherichia coli, is essential for DSF regulation of virulence factor production but not for biofilm dispersal. Deletion of clp in Xcc changed the transcriptional expression of 299 genes including a few encoding transcription factors. Further genetic and microarray analysis led to identification of a homologue of the transcriptional regulator Zur, and a novel TetR‐type transcription factor FhrR. These two regulatory factors regulated different sets of genes within Clp regulon. These results outline a hierarchical signalling network by which DSF modulates different biological functions, and may also provide a clue on how the novel nucleotide signal can be coupled to its downstream regulatory networks.

Succinic semialdehyde couples stress response to quorum-sensing signal decay in Agrobacterium tumefaciens

Quorum sensing (QS) signal decay in Agrobacterium tumefaciens occurs in response to starvation or host signals. We have demonstrated that the γ‐aminobutyric acid (GABA) shunt metabolite links stress response to QS signal decay. Mutation of the aldH gene encoding a succinic semialdehyde dehydrogenase (SSADH) that converts succinic semialdehyde (SSA) to succinic acid results in early expression of the signal degrading enzyme, AttM. Exogenous addition of SSA or its precursor GABA induces AttM expression and abolishes Ti plasmid conjugative transfer. SSA acts by binding to the repressor AttJ that regulates the attKLM operon. attK encodes another SSADH. The stress alarmone ppGpp and SSA modulates separately the expression of the two SSADH enzymes, which might control the intracellular SSA level and hence to switch on/off the QS signal decay system in response to environmental changes. These findings document for the first time a sophisticated signalling mechanism of the widely conserved GABA degradation pathway in prokaryotes.