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Attenuation of virulence in pathogenic bacteria using synthetic quorum-sensing modulators under native conditions on plant hosts.

ABSTRACT: Quorum sensing (QS) is often critical in both pathogenic and mutualistic relationships between bacteria and their eukaryotic hosts. Gram-negative bacteria typically use N-acylated l-homoserine lactone (AHL) signals for QS. We have identified a number of synthetic AHL analogues that are able to strongly modulate QS in culture-based, reporter gene assays. While informative, these assays represent idealized systems, and their relevance to QS under native conditions is often unclear. As one of our goals is to utilize synthetic QS modulators to study bacterial communication under native conditions, identifying robust host-bacteria model systems for their evaluation is crucial. We reasoned that the host-pathogen interaction between Solanum tuberosum (potato) and the Gram-negative pathogen Pectobacterium carotovora would be ideal for such studies as we have identified several potent, synthetic QS modulators for this pathogen, and infection assays in potato are facile. Herein, we report on our development of this host-pathogen system, and another in Phaseolus vulgaris (green bean), as a means for monitoring the ability of abiotic AHLs to modulate QS-regulated virulence in host infection assays. Our assays confirmed that QS modulators previously identified through culture-based assays largely retained their activity profiles when introduced into the plant host. However, inhibition of virulence in wild-type infections was highly dependent on the timing of compound dosing. This study is the first to demonstrate that our AHL analogues are active in wild-type bacteria in their native eukaryotic hosts and provides compelling evidence for the application of these molecules as probes to study QS in a range of organisms and environments.

SUBMITTER: Palmer AG

PROVIDER: S-EPMC3241857 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Attenuation of virulence in pathogenic bacteria using synthetic quorum-sensing modulators under native conditions on plant hosts.

Palmer Andrew G AG Streng Evan E Blackwell Helen E HE

ACS chemical biology 20111006 12

Quorum sensing (QS) is often critical in both pathogenic and mutualistic relationships between bacteria and their eukaryotic hosts. Gram-negative bacteria typically use N-acylated l-homoserine lactone (AHL) signals for QS. We have identified a number of synthetic AHL analogues that are able to strongly modulate QS in culture-based, reporter gene assays. While informative, these assays represent idealized systems, and their relevance to QS under native conditions is often unclear. As one of our g ...[more]

PMID: 21932837

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Project description:Quorum sensing (QS) is a process by which bacteria use low-molecular-weight signaling molecules (or autoinducers) to assess their local population densities and alter gene expression levels at high cell numbers. Many Gram-negative bacteria use N-acyl L-homoserine lactones (AHLs) with aliphatic acyl groups as signaling molecules for QS. However, bacteria that utilize AHLs with aroyl acyl groups have been recently discovered; they include the metabolically versatile soil bacterium Rhodopseudomonas palustris, which uses p-coumaroyl HL (p-cAHL) as its QS signal. This autoinducer is especially unusual because its acyl group is believed to originate from a monolignol (i.e., p-coumarate) produced exogenously by plants in the R. palustris environment, rather than through the endogenous fatty acid biosynthesis pathway like other native AHLs. As such, p-cAHL could signal not only bacterial density, but also the availability of an exogenous plant-derived substrate and might even constitute an interkingdom signal. Like other Gram-negative bacteria, QS in R. palustris is controlled by the p-cAHL signal binding its cognate LuxR-type receptor, RpaR. We sought to determine if non-native aroyl HLs (ArHLs) could potentially activate or inhibit RpaR in R. palustris, and thereby modulate QS in this bacterium. Herein, we report the testing of a set of synthetic ArHLs for RpaR agonism and antagonism by using a R. palustris reporter strain. Several potent non-native RpaR agonists and antagonists were identified. Additionally, the screening data revealed that lower concentrations of ArHL are required to strongly agonize RpaR than to antagonize it. Structure-activity relationship analyses of the active ArHLs indicated that potent RpaR agonists tend to have sterically small substituents on their aryl groups, most notably in the ortho position. In turn, the most potent RpaR antagonists were based on either the phenylpropionyl HL (PPHL) or the phenoxyacetyl HL (POHL) scaffold, and many contained an electron-withdrawing group at either the meta or para positions of the aryl ring. To our knowledge, the compounds reported herein represent the first abiotic chemical modulators of RpaR, and more generally, the first abiotic ligands capable of intercepting QS in bacteria that utilize native ArHL signals. In view of the origins of the p-cAHL signal in R. palustris, the largely unknown role of QS in this bacterium, and R. palustris' unique environmental lifestyles, we anticipate that these compounds could be valuable as chemical probes to study QS in R. palustris in a range of fundamental and applied contexts.

Project description:Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH?) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

Dataset Information

Attenuation of virulence in pathogenic bacteria using synthetic quorum-sensing modulators under native conditions on plant hosts.

Publications

Attenuation of virulence in pathogenic bacteria using synthetic quorum-sensing modulators under native conditions on plant hosts.

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