Jin-Ling Xu

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.

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species

ABSTRACT A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.

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.

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.

The Acyl-Homoserine Lactone-Type Quorum-Sensing System Modulates Cell Motility and Virulence of Erwinia chrysanthemi pv. zeae

Erwinia chrysanthemi pv. zeae is one of the Erwinia chrysanthemi pathovars that infects on both dicotyledons and monocotyledons. However, little is known about the molecular basis and regulatory mechanisms of its virulence. By using a transposon mutagenesis approach, we cloned the genes coding for an E. chrysanthemi pv. zeae synthase of acyl-homoserine lactone (AHL) quorum-sensing signals (expI(Ecz)) and a cognate response regulator (expR(Ecz)). Chromatography analysis showed that expI(Ecz) encoded production of the AHL signal N-(3-oxo-hexanoyl)-homoserine lactone (OHHL). Null mutation of expI(Ecz) in the E. chrysanthemi pv. zeae strain EC1 abolished AHL production, increased bacterial swimming and swarming motility, disabled formation of multicell aggregates, and attenuated virulence of the pathogen on potato tubers. The mutation also marginally reduced the inhibitory activity of E. chrysanthemi pv. zeae on rice seed germination. The mutant phenotypes were rescued by either exogenous addition of AHL signal or in trans expression of expI(Ecz). These data demonstrate that the AHL-type QS signal plays an essential role in modulation of E. chrysanthemi pv. zeae cell motility and the ability to form multicell aggregates and is involved in regulation of bacterial virulence.

VqsM, a novel AraC‐type global regulator of quorum‐sensing signalling and virulence in Pseudomonas aeruginosa

Human pathogen Pseudomonas aeruginosa uses quorum‐sensing (QS) signalling systems to synchronize the production of virulence factors. There are two interrelated QS systems, las and rhl, in P. aeruginosa. In addition to this complexity, a number of transcriptional regulators were shown to have complicated interplays with las and rhl central QS components. Here, we describe a novel virulence and QS modulator (VqsM) that positively regulates the QS systems in P. aeruginosa. Mutation in vqsM resulted in much reduced production of N‐acylhomoserine lactones (AHLs) and extracellular enzymes. Sequence analysis revealed that vqsM encodes a transcriptional regulator with an AraC‐type helix–turn–helix DNA binding domain at the C‐terminal of the peptide. Global gene expression profile analysis showed at least a total of 302 genes to be influenced, directly or indirectly, by VqsM. Among the 203 VqsM‐promoted genes, 52.2% were known to be QS upregulated. Several genes encoding the key regulators implicated in QS, such as rhlR, rsaL, vqsR, mvfR, pprB and rpoS, and two AHL synthesis genes, lasI and rhlI, were suppressed in the vqsM mutant. Similar to the ‘AHL‐blind’ phenotype of vqsR and pprB mutants, vqsM mutant did not respond to external addition of N‐3‐oxo‐dodecanoyl‐homoserine lactone signals. Moreover, overexpression of vqsR in vqsM mutant more or less restored the production of both AHL and virulence factors. The results demonstrate that VqsM, largely through modulation of vqsR expression, plays a vital role in regulation of QS signalling in P. aeruginosa.

Molecular and conformational basis of a specific and high-affinity interaction between AlbA and albicidin phytotoxin

ABSTRACT The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 × 10−8 M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys106, Trp110, Tyr113, Leu114, Tyr126, Pro134, and Trp162 with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His125, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.