Quorum Quenching in Culturable Phyllosphere Bacteria from Tobacco

Many Gram-negative plant pathogenic bacteria employ a N-acylhomoserine lactone (AHL)-based quorum sensing (QS) system to regulate their virulence traits. A sustainable biocontrol strategy has been developed using quorum quenching (QQ) bacteria to interfere with QS and protect plants from pathogens. Here, the prevalence and the diversity of QQ strains inhabiting tobacco leaf surfaces were explored. A total of 1177 leaf-associated isolates were screened for their ability to disrupt AHL-mediated QS, using the biosensor Chromobacterium violaceum CV026. One hundred and sixty-eight strains (14%) are capable of interfering with AHL activity. Among these, 106 strains (63%) of the culturable quenchers can enzymatically degrade AHL molecules, while the remaining strains might use other QS inhibitors to interrupt the chemical communication. Moreover, almost 79% of the QQ strains capable of inactivating AHLs enzymatically have lactonase activity. Further phylogenetic analysis based on 16S rDNA revealed that the leaf-associated QQ bacteria can be classified as Bacillus sp., Acinetobacter sp., Lysinibacillus sp., Serratia sp., Pseudomonas sp., and Myroides sp. The naturally occurring diversity of bacterial quenchers might provide opportunities to use them as effective biocontrol reagents for suppressing plant pathogen in situ.     

基于群体淬灭理论MBR减缓膜污染研究进展

膜生物反应器(membrane bioreactor,MBR)已成为一个十分有前景的废水处理工艺,该工艺具有出水水质好、占地面积小等优势.然而,由于微生物附着在膜表面而发生的生物污染现象,致使出水通量下降,限制了MBR的大规模推广应用.最近,一种新兴的、基于群体感应(quorum sensing,QS)的群体淬灭(quorum quenching,QQ)技术在延缓MBR膜污染领域的应用备受关注.QQ通过干扰群体感应系统而阻止其所依赖信号分子的基因表达,从而可有效抑制膜表面生物膜的形成.本文首先介绍了群体感应机理,并根据不同的群体淬灭物质,归纳总结了群体淬灭技术在延缓MBR膜污染的最新研究进展,最后对该领域的研究进行了展望.     

Quorum sensing in bacterial species that use degenerate autoinducers can be tuned by using structurally identical non-native ligands

Many bacteria use quorum sensing (QS) to regulate cell-density dependent phenotypes that play critical roles in the maintenance of their associations with eukaryotic hosts. In Gram-negative bacteria, QS is primarily controlled by N-acylated L-homoserine lactone (AHL) signals and their cognate LuxR-type receptors. AHL-LuxR-type receptor binding regulates the expression of target genes necessary for QS phenotypes. We recently identified a series of non-native AHLs capable of intercepting AHL-LuxR binding in the marine symbiont Vibrio fischeri, and thereby strongly promoting or inhibiting QS in this organism. V. fischeri utilizes N-(3-oxo)-hexanoyl L-HL (OHHL) as its primary QS signal, and OHHL is also used by several other bacterial species for QS. Such signal degeneracy is common among bacteria, and we sought to determine if our non-native LuxR agonists and antagonists, which are active in V. fischeri, would also modulate QS phenotypes in other bacteria that use OHHL. Herein, we report investigations into the activity of a set of synthetic LuxR modulators in the plant pathogen Pectobacterium carotovora subsp. carotovora Ecc71. This pathogen uses OHHL and two closely related LuxR-type receptors, ExpR1 and ExpR2, to control virulence, and we evaluated their responses to synthetic ligands by quantifying virulence factor production. Our results suggest an overall conservation in the activity trends of the ligands between the ExpR receptors in P. carotovora Ecc71 and LuxR in V. fischeri, and indicate that these compounds could be used as tools to study QS in an expanded set of bacteria. Notable differences in activity were apparent for certain compounds, however, and suggest that it might be possible to selectively regulate QS in bacteria that utilize degenerate AHLs.     

Nano-Formulation Endows Quorum Quenching Enzyme-Antibiotic Hybrids with Improved Antibacterial and Antibiofilm Activities against Pseudomonas aeruginosa

The emergence of antibiotic resistant bacteria coupled with the shortage of efficient antibacterials is one of the most serious unresolved problems for modern medicine. In this study, the nano-hybridization of the clinically relevant antibiotic, gentamicin, with the bacterial pro-pathological cell-to-cell communication-quenching enzyme, acylase, is innovatively employed to increase its antimicrobial efficiency against Pseudomonas aeruginosa planktonic cells and biofilms. The sonochemically generated hybrid gentamicin/acylase nano-spheres (GeN_AC NSs) showed a 16-fold improved bactericidal activity when compared with the antibiotic in bulk form, due to the enhanced physical interaction and disruption of the P. aeruginosa cell membrane. The nano-hybrids attenuated 97 ± 1.8% of the quorum sensing-regulated virulence factors’ production and inhibited the bacterium biofilm formation in an eight-fold lower concentration than the stand-alone gentamicin NSs. The P. aeruginosa sensitivity to GeN_AC NSs was also confirmed in a real time assay monitoring the bacterial cells elimination, using a quartz crystal microbalance with dissipation. In protein-enriched conditions mimicking the in vivo application, these hybrid nano-antibacterials maintained their antibacterial and antibiofilm effectiveness at concentrations innocuous to human cells. Therefore, the novel GeN_AC NSs with complementary modes of action show potential for the treatment of P. aeruginosa biofilm infections at a reduced antibiotic dosage.     

Identification of FadT as a Novel Quorum Quenching Enzyme for the Degradation of Diffusible Signal Factor in Cupriavidus pinatubonensis Strain HN-2

Quorum sensing (QS) is a microbial cell–cell communication mechanism and plays an important role in bacterial infections. QS-mediated bacterial infections can be blocked through quorum quenching (QQ), which hampers signal accumulation, recognition, and communication. The pathogenicity of numerous bacteria, including Xanthomonas campestris pv. campestris (Xcc), is regulated by diffusible signal factor (DSF), a well-known fatty acid signaling molecule of QS. Cupriavidus pinatubonensis HN-2 could substantially attenuate the infection of XCC through QQ by degrading DSF. The QQ mechanism in strain HN-2, on the other hand, is yet to be known. To understand the molecular mechanism of QQ in strain HN-2, we used whole-genome sequencing and comparative genomics studies. We discovered that the fadT gene encodes acyl-CoA dehydrogenase as a novel QQ enzyme. The results of site-directed mutagenesis demonstrated the requirement of fadT gene for DSF degradation in strain HN-2. Purified FadT exhibited high enzymatic activity and outstanding stability over a broad pH and temperature range with maximal activity at pH 7.0 and 35 °C. No cofactors were required for FadT enzyme activity. The enzyme showed a strong ability to degrade DSF. Furthermore, the expression of fadT in Xcc results in a significant reduction in the pathogenicity in host plants, such as Chinese cabbage, radish, and pakchoi. Taken together, our results identified a novel DSF-degrading enzyme, FadT, in C. pinatubonensis HN-2, which suggests its potential use in the biological control of DSF-mediated pathogens.     

群体感应抑制控制膜生物污染的研究进展

水中微生物及其代谢产物沉积吸附在膜表面,进而生长繁殖,形成了膜生物污染。膜生物污染的控制已经成为学者们研究的热点之一,而基于驱散群体感应体系对膜生物污染进行控制是国内外新兴的研究课题之一。本文综述了传统的膜生物污染控制方法,并阐述了基于驱散群体感应体系控制膜生物污染的研究新进展,并提出前景展望。     

A Genetic Circuit System Based on Quorum Sensing Signaling for Directed Evolution of Quorum‐Quenching Enzymes

Quorum sensing is a cell–cell communication mechanism that is involved in the regulation of biological functions such as luminescence, virulence, and biofilm formation. Quorum‐quenching enzymes, which interrupt quorum‐sensing signaling through degradation of quorum‐sensing molecules, have emerged as a new approach to controlling and preventing bacterial virulence and pathogenesis. In an effort to develop quorum‐quenching enzymes with improved catalytic activities, a genetic circuit system based on acylhomoserine‐lactone (AHL)‐mediated quorum‐sensing signaling was constructed. The genetic circuit system was composed of lux‐R, lux‐I promoter, β‐lactamase, and β‐lactamase inhibitor, and designed to confer antibiotic resistance on host cells expressing an AHL‐degrading enzyme, thereby enabling rapid screening of quorum‐quenching enzymes. To demonstrate the utility of the genetic circuit system, we attempted the directed evolution of the AHL hydrolase from Bacillus sp. The genetic circuit system was shown to be effective in screening of quorum‐quenching enzymes with high catalytic efficiency. From these results it is expected that the genetic circuit system can be widely used for the isolation and directed evolution of quorum‐quenching enzymes with greater potential.     

Potent and selective synthetic modulators of a quorum sensing repressor in Pseudomonas aeruginosa identified from second-generation libraries of N-acylated L-homoserine lactones

Bacteria can coordinate group behavior using chemical signals in a process called quorum sensing (QS). The QS system in the opportunistic pathogen Pseudomonas aeruginosa is largely governed by the LasR receptor and its cognate chemical signal, N-(3-oxo)-dodecanoyl L-homoserine lactone (OdDHL). LasR also appears to share this signal with an orphan LuxR-type receptor in P. aeruginosa, termed QscR, which represses LasR activity. Non-native molecules that modulate QscR would represent valuable tools to study the role of this novel QS repressor protein in P. aeruginosa. We performed a critical analysis of previously identified, non-native N-acylated L-homoserine lactone (AHL) activators and inhibitors of QscR to determine a set of structure-activity relationships (SARs). Based on these SAR data, we designed, synthesized, and screened several second-generation libraries of AHLs for new ligands that could target QscR. These studies revealed the most active AHL agonists and antagonists of QscR reported to date, with activities ranging from nanomolar to low micromolar in a QscR bacterial reporter strain. Several of these AHLs were highly selective for QscR over LasR and other LuxR-type receptors. A small subset of the new QscR activators, however, were also found to inhibit LasR; this demonstrates the exciting potential for the synergistic modulation of these integral P. aeruginosa QS receptors by using a single synthetic compound.     

Choosing an Appropriate Infection Model to Study Quorum Sensing Inhibition in Pseudomonas Infections

Bacteria, although considered for decades to be antisocial organisms whose sole purpose is to find nutrients and multiply are, in fact, highly communicative organisms. Referred to as quorum sensing, cell-to-cell communication mechanisms have been adopted by bacteria in order to co-ordinate their gene expression. By behaving as a community rather than as individuals, bacteria can simultaneously switch on their virulence factor production and establish successful infections in eukaryotes. Understanding pathogen-host interactions requires the use of infection models. As the use of rodents is limited, for ethical considerations and the high costs associated with their use, alternative models based on invertebrates have been developed. Invertebrate models have the benefits of low handling costs, limited space requirements and rapid generation of results. This review presents examples of such models available for studying the pathogenicity of the Gram-negative bacterium Pseudomonas aeruginosa. Quorum sensing interference, known as quorum quenching, suggests a promising disease-control strategy since quorum-quenching mechanisms appear to play important roles in microbe-microbe and host-pathogen interactions. Examples of natural and synthetic quorum sensing inhibitors and their potential as antimicrobials in Pseudomonas-related infections are discussed in the second part of this review.     

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.     

Isolation and Molecular Characterization of Biofouling Bacteria and Profiling of Quorum Sensing Signal Molecules from Membrane Bioreactor Activated Sludge

The formation of biofilm in a membrane bioreactor depends on the production of various signaling molecules like N-acyl homoserine lactones (AHLs). In the present study, a total of 200 bacterial strains were isolated from membrane bioreactor activated sludge and screened for AHLs production using two biosensor systems, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136. A correlation between AHLs production and biofilm formation has been made among screened AHLs producing strains. The 16S rRNA gene sequence analysis revealed the dominance of Aeromonas and Enterobacter sp. in AHLs production; however few a species of Serratia, Leclercia, Pseudomonas, Klebsiella, Raoultella and Citrobacter were also identified. The chromatographic characterization of sludge extract showed the presence of a broad range of quorum sensing signal molecules. Further identification of sludge AHLs by thin layer chromatography bioassay and high performance liquid chromatography confirms the presence of C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, C12-HSL, 3-oxo-C12-HSL and C14-HSL. The occurrence of AHLs in sludge extract and dominance of Aeromonas and Enterobacter sp. in activated sludge suggests the key role of these bacterial strains in AHLs production and thereby membrane fouling.     

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.     

Interkingdom Responses to Bacterial Quorum Sensing Signals Regulate Frequency and Rate of Nodulation in Legume–Rhizobia Symbiosis

Density‐dependent phenotypic switching in bacteria, the phenomenon of quorum sensing (QS), is instrumental in many pathogenic and mutualistic behaviors. In many Gram‐negative bacteria, QS is regulated by N‐acylated‐l ‐homoserine lactones (AHLs). Synthetic analogues of these AHLs hold significant promise for regulating QS at the host–symbiont interface. Regulation depends on refined temporal and spatial models of quorums under native conditions. Critical to this is an understanding of how the presence of these signals may affect a prospective host. We screened a library of AHL analogues for their ability to regulate the legume–rhizobia mutualistic symbiosis (nodulation) between Medicago truncatula and Sinorhizobium meliloti. Using an established QS‐reporter line of S. meliloti and nodulation assays with wild‐type bacteria, we identified compounds capable of increasing either the rate of nodule formation or total nodule number. Most importantly, we identified compounds with activity exclusive to either host or pathogen, underscoring the potential to generate QS modulators selective to bacteria with limited effects on a prospective host.     

A Nitric Oxide-Responsive Quorum Sensing Circuit in Vibrio harveyi Regulates Flagella Production and Biofilm Formation

Cell signaling plays an important role in the survival of bacterial colonies. They use small molecules to coordinate gene expression in a cell density dependent manner. This process, known as quorum sensing, helps bacteria regulate diverse functions such as bioluminescence, biofilm formation and virulence. In Vibrio harveyi, a bioluminescent marine bacterium, four parallel quorum-sensing systems have been identified to regulate light production. We have previously reported that nitric oxide (NO), through the H-NOX/HqsK quorum sensing pathway contributes to light production in V. harveyi through the LuxU/LuxO/LuxR quorum sensing pathway. In this study, we show that nitric oxide (NO) also regulates flagellar production and enhances biofilm formation. Our data suggest that V. harveyi is capable of switching between lifestyles to be able to adapt to changes in the environment.     

The Role of Antibodies in the Treatment of SARS-CoV-2 Virus Infection,and Evaluating Their Contribution to Antibody-Dependent Enhancement of Infection

Antibodies play a crucial role in the immune response, in fighting off pathogens as well as helping create strong immunological memory. Antibody-dependent enhancement (ADE) occurs when non-neutralising antibodies recognise and bind to a pathogen, but are unable to prevent infection, and is widely known and is reported as occurring in infection caused by several viruses. This narrative review explores the ADE phenomenon, its occurrence in viral infections and evaluates its role in infection by SARS-CoV-2 virus, which causes coronavirus disease 2019 (COVID-19). As of yet, there is no clear evidence of ADE in SARS-CoV-2, though this area is still subject to further study.     

Fabrication of Porous TiO<Subscript>2</Subscript> Hollow Spheres and Their Application in Gas Sensing

In this work, porous TiO₂ hollow spheres with an average diameter of 100 nm and shell thickness of 20 nm were synthesized by a facile hydrothermal method with NH₄HCO₃ as the structure-directing agent, and the formation mechanism for this porous hollow structure was proved to be the Ostwald ripening process by tracking the morphology of the products at different reaction stages. The product was characterized by SEM, TEM, XRD and BET analyses, and the results show that the as-synthesized products are anatase phase with a high surface area up to 132.5 m²/g. Gas-sensing investigation reveals that the product possesses sensitive response to methanal gas at 200°C due to its high surface area.