Fine‐Tuning Covalent Inhibition of Bacterial Quorum Sensing

Emerging antibiotic resistance among human pathogens has galvanized efforts to find alternative routes to combat bacterial virulence. One new approach entails interfering with the ability of bacteria to coordinate population‐wide gene expression, or quorum sensing (QS), thus inhibiting the production of virulence factors and biofilm formation. We have recently developed such a strategy by targeting LasR, the master regulator of QS in the opportunistic human pathogen Pseudomonas aeruginosa, through the rational design of covalent inhibitors closely based on the core structure of the native ligand. We now report several groups of new inhibitors, one of which, fluoro‐substituted ITC‐12, displayed complete covalent modification of LasR, as well as effective QS inhibition in vitro and promising in vivo results. In addition to their potential clinical relevance, this series of synthetic QS modulators can be used as a tool to further unravel the complicated QS regulation in P. aeruginosa.     

Non‐native N‐Aroyl L‐Homoserine Lactones Are Potent Modulators of the Quorum Sensing Receptor RpaR in Rhodopseudomonas palustris

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.     

Synthesis and Evaluation of New Antagonists of Bacterial Quorum Sensing in Vibrio harveyi

Bacterial quorum sensing has received much attention in recent years because of its relevance to pathological events such as biofilm formation. Based on the structures of two lead inhibitors (IC₅₀: 35–55 μM ) against autoinducer‐2‐mediated quorum sensing identified through virtual screening, we synthesized 39 analogues and examined their inhibitory activities. Twelve of these new analogues showed equal or better inhibitory activities than the lead inhibitors. The best compound showed an IC₅₀ value of ～6 μM in a whole‐cell assay using Vibrio harveyi as the model organism. The structure–activity relationship is discussed herein.     

Active Efflux Influences the Potency of Quorum Sensing Inhibitors in Pseudomonas aeruginosa

Many bacteria regulate gene expression through a cell–cell signaling process called quorum sensing (QS). In proteobacteria, QS is largely mediated by signaling molecules known as N‐acylated L ‐homoserine lactones (AHLs) and their associated intracellular LuxR‐type receptors. The design of non‐native small molecules capable of inhibiting LuxR‐type receptors (and thereby QS) in proteobacteria is an active area of research, and numerous lead compounds are AHL derivatives that mimic native AHL molecules. Much of this previous work has focused on the pathogen Pseudomonas aeruginosa, which controls an arsenal of virulence factors and biofilm formation through QS. The MexAB‐OprM efflux pump has been shown to play a role in the secretion of the major AHL signal in P. aeruginosa, N‐(3‐oxododecanoyl) L ‐homoserine lactone. In the current study, we show that a variety of non‐native AHLs and related derivatives capable of inhibiting LuxR‐type receptors in P. aeruginosa display significantly higher potency in a P. aeruginosa Δ(mexAB‐oprM) mutant, suggesting that MexAB‐OprM also recognizes these compounds as substrates. We also demonstrate that the potency of 5,6‐dimethyl‐2‐aminobenzimidazole, recently shown to be a QS and biofilm inhibitor in P. aeruginosa, is not affected by the presence/absence of the MexAB‐OprM pump. These results have implications for the use of non‐native AHLs and related derivatives as QS modulators in P. aeruginosa and other bacteria, and provide a potential design strategy for the development of new QS modulators that are resistant to active efflux.     

Solid‐Phase Synthesis and Biological Evaluation of N‐Dipeptido L‐Homoserine Lactones as Quorum Sensing Activators

Bacteria use small signaling molecules to communicate in a process termed “quorum sensing” (QS), which enables the coordination of survival strategies, such as production of virulence factors and biofilm formation. In Gram‐negative bacteria, these signaling molecules are a series of N‐acylated L ‐homoserine lactones. With the goal of identifying non‐native compounds capable of modulating bacterial QS, a virtual library of N‐dipeptido L ‐homoserine lactones was screened in silico with two different crystal structures of LasR. The 30 most promising hits were synthesized on HMBA‐functionalized PEGA resin and released through an efficient acid‐mediated cyclative release mechanism. Subsequent screening for modulation of QS in Pseudomonas aeruginosa and E. coli identified six moderately strong activators. A follow‐up library designed from the preliminary derived structure–activity relationships was synthesized and evaluated for their ability to activate the QS system in this bacterium. This resulted in the identification of another six QS activators (two with low micromolar activity) thus illuminating structural features required for QS modulation.     

Complementary Regulation of BfmRS Two-Component and AbaIR Quorum Sensing Systems to Express Virulence-Associated Genes in Acinetobacter baumannii

Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The ΔbfmS mutant exhibited a significant decrease in surface motility, which presumably resulted from the low expression of pilT and A1S_0112-A1S_0119 gene cluster. The ΔbfmR mutant displayed a significant reduction in biofilm and pellicle formation due to the low expression of csu operon. The deletion of abaR did not affect the expression of bfmR or bfmS. However, the expression of abaR and abaI was upregulated in the ΔbfmR mutant. The ΔbfmR mutant also produced more autoinducers than did the wild-type strain, suggesting that BfmR negatively regulates the AbaIR QS system. The ΔbfmS mutant exhibited no autoinducer production in the bioassay system. The expression of the A1S_0112-A1S_0119 gene cluster was downregulated in the ΔabaR mutant, whereas the expression of csu operon was upregulated in this mutant with a high cell density. In conclusion, for the first time, we demonstrated that the BfmRS-AbaIR QS system axis regulated the expression of virulence-associated genes in A. baumannii. This study provides new insights into the complex network system involved in the regulation of virulence-associated genes underlying the pathogenicity of A. baumannii.     

Identification,Quantification, and Determination of the Absolute Configuration of the Bacterial Quorum‐Sensing Signal Autoinducer‐2 by Gas Chromatography–Mass Spectrometry

Sensing the signal : A gas chromatography–mass spectrometry (GC–MS) method for the analysis of the quorum‐sensing autoinducer‐2 is described. It allows, for the first time, the direct analysis and accurate determination of this highly water soluble signaling compound, which exists in complex equilibria. The application on the caries‐causing bacterium Streptococcus mutans is described.

     

Potent and Selective Modulation of the RhlR Quorum Sensing Receptor by Using Non‐native Ligands: An Emerging Target for Virulence Control in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses N‐acylated l ‐homoserine lactone signals and a triumvirate of LuxR‐type receptor proteins—LasR, RhlR, and QscR—for quorum sensing (QS). Each of these receptors can contribute to QS activation or repression and, thereby, the control of myriad virulence phenotypes in this pathogen. LasR has traditionally been considered to be at the top of the QS receptor hierarchy in P. aeruginosa; however, recent reports suggest that RhlR plays a more prominent role in infection than originally predicted, in some circumstances superseding that of LasR. Herein, we report the characterization of a set of synthetic, small‐molecule agonists and antagonists of RhlR. Using E. coli reporter strains, we demonstrated that many of these compounds can selectively activate or inhibit RhlR instead of LasR and QscR. Moreover, several molecules maintain their activities in P. aeruginosa at concentrations analogous to native RhlR signal levels. These compounds represent useful chemical probes to study the role of RhlR in the complex QS circuitry of P. aeruginosa, its direct (and indirect) effects on virulence, and its overall merit as a target for anti‐infective therapy.     

Biosynthesis and the Roles of Plant Sterols in Development and Stress Responses

Plant sterols are important components of the cell membrane and lipid rafts, which play a crucial role in various physiological and biochemical processes during development and stress resistance in plants. In recent years, many studies in higher plants have been reported in the biosynthesis pathway of plant sterols, whereas the knowledge about the regulation and accumulation of sterols is not well understood. In this review, we summarize and discuss the recent findings in the field of plant sterols, including their biosynthesis, regulation, functions, as well as the mechanism involved in abiotic stress responses. These studies provide better knowledge on the synthesis and regulation of sterols, and the review also aimed to provide new insights for the global role of sterols, which is liable to benefit future research on the development and abiotic stress tolerance in plant.     

Roles of Plant Glycine-Rich RNA-Binding Proteins in Development and Stress Responses

In recent years, much progress has been made in elucidating the functional roles of plant glycine-rich RNA-binding proteins (GR-RBPs) during development and stress responses. Canonical GR-RBPs contain an RNA recognition motif (RRM) or a cold-shock domain (CSD) at the N-terminus and a glycine-rich domain at the C-terminus, which have been associated with several different RNA processes, such as alternative splicing, mRNA export and RNA editing. However, many aspects of GR-RBP function, the targeting of their RNAs, interacting proteins and the consequences of the RNA target process are not well understood. Here, we discuss recent findings in the field, newly defined roles for GR-RBPs and the actions of GR-RBPs on target RNA metabolism.