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With the increasing resistance exhibited by undesirable bacteria to traditional antibiotics, the need to discover alternative (or, at least, supplementary) treatments to combat chemically resistant bacteria is becoming urgent. Quorum sensing (QS) refers to a novel bacterial communication system for monitoring cell density and regulation of a network of gene expression that is mediated by a group of signaling molecules called autoinducers (AIs). QS-regulated multicellular behaviors include biofilm formation, horizontal gene transfer, and antibiotic synthesis, which are demonstrating increasing pathogenicity to plants and aquacultural animals as well as contamination of wastewater treatment devices. To inhibit QS-regulated microbial behaviors, the strategy of quorum quenching (QQ) has been developed. Different quorum quenchers interfere with QS through different mechanisms, such as competitively inhibiting AI perception (e.g., by QS inhibitors) and AI degradation (e.g., by QQ enzymes). In this review, we first introduce different signaling molecules, including diffusible signal factor (DSF) and acyl homoserine lactones (AHLs) for Gram-negative bacteria, AIPs for Gram-positive bacteria, and AI-2 for interspecies communication, thus demonstrating the mode of action of the QS system. We next exemplify the QQ mechanisms of various quorum quenchers, such as chemical QS inhibitors, and the physical/enzymatic degradation of QS signals. We devote special attention to AHL-degrading enzymes, which are categorized in detail according to their diverse catalytic mechanisms and enzymatic properties. In the final part, the applications and advantages of quorum quenchers (especially QQ enzymes and bacteria) are summarized in the context of agricultural/aquacultural pathogen biocontrol, membrane bioreactors for wastewater treatment, and the attenuation of human pathogenic bacteria. Taken together, we present the state-of-the-art in research considering QS and QQ, providing theoretical evidence and support for wider application of this promising environmentally friendly biocontrol strategy.

Vast quantities of synthetic pesticides have been widely applied in various fields to kill plant pathogens, resulting in increased pathogen resistance and decreased effectiveness of such chemicals. In addition, the increased presence of pesticide residues affects living organisms and the environment largely on a global scale. To mitigate the impact of crop diseases more sustainably on plant health and productivity, there is a need for more safe and more eco-friendly strategies as compared to chemical prevention. Quorum sensing (QS) is an intercellular communication mechanism in a bacterial population, through which bacteria adjust their population density and behavior upon sensing the levels of signaling molecules in the environment. As an alternative, quorum quenching (QQ) is a promising new strategy for disease control, which interferes with QS by blocking intercellular communication between pathogenic bacteria to suppress the expression of disease-causing genes. Black rot caused by Xanthomonas campestris pv. campestris ( Xcc ) is associated with the diffusible signal factor (DSF). As detailed in this study, a new QQ strain F25, identified as Burkholderia sp., displayed a superior ability to completely degrade 2 mM of DSF within 72 h. The main intermediate product in the biodegradation of DSF was identified as n-decanoic acid, based on gas chromatography-mass spectrometry (GC-MS). A metabolic pathway for DSF by strain F25 is proposed, based on the chemical structure of DSF and its intermediates, demonstrating the possible degradation of DSF via oxidation-reduction. The application of strain F25 and its crude enzyme as biocontrol agents significantly attenuated black rot caused by Xcc , and inhibited tissue maceration in the host plant Raphanus sativus L., without affecting the host plant. This suggests that agents produced from strain F25 and its crude enzyme have promising applications in controlling infectious diseases caused by DSF-dependent bacterial pathogens. These findings are expected to provide a new therapeutic strategy for controlling QS-mediated plant diseases.

Bacteria and their eukaryotic hosts have co-evolved for millions of years, and the former can intercept eukaryotic signaling systems for the successful colonization of the host. The diffusible signal factor (DSF) family represents a type of quorum-sensing signals found in diverse Gram-negative bacterial pathogens. Recent evidence shows that the DSF is involved in interkingdom communications between the bacterial pathogen and the host plant. In this study, we explored the anti-inflammatory effect of the DSF and its underlying molecular mechanism in a zebrafish model. We found that the DSF treatment exhibited a strong protective effect on the inflammatory response of zebrafish induced by lipopolysaccharide (LPS). In the LPS-induced inflammation zebrafish model, the DSF could significantly ameliorate the intestinal pathological injury, reduce abnormal migration and the aggregation of inflammatory cells, inhibit the excessive production of inflammatory mediator reactive oxygen species (ROS) content, and prevent apoptosis. Through an RNA-Seq analysis, a total of 938 differentially expressed genes (DEGs) was screened between LPS and LPS + DSF treatment zebrafish embryos. A further bioinformatics analysis and validation revealed that the DSF might inhibit the LPS-induced zebrafish inflammatory response by preventing the activation of signaling in the Toll-like receptor pathway, attenuating the expression of pro-inflammatory cytokines and chemokines, and regulating the activation of the caspase cascade through restoring the expression of lysosomal cathepsins and apoptosis signaling. This study, for the first time, demonstrates the anti-inflammatory role and a potential pharmaceutical application of the bacterial signal DSF. These findings also suggest that the interkingdom communication between DSF-producing bacteria and zebrafish might occur in nature.

Abstract Xanthomonas is an important genus of plant-associated bacteria that causes significant yield losses of economically important crops worldwide. Different approaches have assessed genetic diversity and evolutionary interrelationships among the Xanthomonas species. However, information from clustered regularly interspaced short palindromic repeats (CRISPRs) has yet to be explored. In this work, we analyzed the architecture of CRISPR–Cas loci and presented a sequence similarity-based clustering of conserved Cas proteins in different species of Xanthomonas. Although absent in many investigated genomes, Xanthomonas harbors subtype I-C and I-F CRISPR–Cas systems. The most represented species, Xanthomonas citri, presents a great diversity of genome sequences with an uneven distribution of the CRISPR–Cas systems among the subspecies/pathovars. Only X. citri subsp. citri and X. citri pv. punicae have these systems, exclusively of subtype I-C system. Moreover, the most likely targets of the X. citri CRISPR spacers are viruses (phages). At the same time, few are plasmids, indicating that CRISPR/Cas system is possibly a mechanism to control the invasion of foreign DNA. We also showed in X. citri susbp. citri that the cas genes are regulated by the diffusible signal factor, the quorum sensing (QS) signal molecule, according to cell density increases, and under environmental stress like starvation. These results suggest that the regulation of CRISPR–Cas by QS occurs to activate the gene expression only during phage infection or due to environmental stresses, avoiding a possible reduction in fitness. Although more studies are needed, CRISPR–Cas systems may have been selected in the Xanthomonas genus throughout evolution, according to the cost-benefit of protecting against biological threats and fitness maintenance in challenging conditions. The presence and distribution of clustered regularly interspaced short palindromic repeat-associated sequence loci in Xanthomonas, with potencial regulatation by the quorum sensing mechanism.

Background The gram-negative Xanthomonas genus contains a large group of economically important plant pathogens, which cause severe diseases on many crops worldwide. The diffusible signal factor (DSF) - mediated quorum sensing (QS) system coordinates expression of virulence factors in plant pathogenic Xanthomonas spp. However, the regulatory effects of this system during the Xanthomonas- plant interactions remain unclear from both the pathogen and host aspects. Results In this study, we investigated the in planta DSF- mediated QS regulon of X. citri subsp . citri ( Xac ), the causal agent of citrus canker. We also characterized the transcriptional responses of citrus plants to DSF-mediated Xac infection via comparing the gene expression patterns of citrus trigged by wild type Xac strain 306 with those trigged by its DSF- deficient (∆ rpfF ) mutant using the dual RNA-seq approach. Comparative global transcript profiles of Xac strain 306 and the ∆ rpfF mutant during host infection revealed that DSF- mediated QS specifically modulates bacterial adaptation, nutrition uptake and metabolisms, stress tolerance, virulence, and signal transduction to favor host infection. The transcriptional responses of citrus to DSF-mediated Xac infection are characterized by downregulation of photosynthesis genes and plant defense related genes, suggesting photosynthetically inactive reactions and repression of defense responses. Alterations of phytohormone metabolism and signaling pathways were also triggered by DSF-mediated Xac infection to benefit the pathogen. Conclusions Collectively, our findings provide new insight into the DSF- mediated QS regulation during plant-pathogen interactions and advance the understanding of traits used by Xanthomonas to promote infection on host plants.

Quorum sensing is a type of chemical communication by which bacterial populations control expression of their genes in a coordinated manner. This regulatory mechanism is commonly used by pathogens to control the expression of genes encoding virulence factors and that of genes involved in the bacterial adaptation to variations in environmental conditions. In phytopathogenic bacteria, several mechanisms of quorum sensing have been characterized. In this review, we describe the different quorum sensing systems present in phytopathogenic bacteria, such as those using the signal molecules named N-acyl-homoserine lactone (AHL), diffusible signal factor (DSF), and the unknown signal molecule of the virulence factor modulating (VFM) system. We focus on studies performed on phytopathogenic bacteria of major importance, including Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella,Dickeya, and Pectobacterium spp. For each system, we present the mechanism of regulation, the functions targeted by the quorum sensing system, and the mechanisms by which quorum sensing is regulated.

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.

The diffusible signal factor (DSF) is a fatty acid signal molecule and is widely conserved in various Gram-negative bacteria. DSF is involved in the regulation of pathogenic virulence in many bacterial pathogens, including Xanthomonas campestris pv. campestris (Xcc). Quorum quenching (QQ) is a potential approach for preventing and controlling DSF-mediated bacterial infections by the degradation of the DSF signal. Acinetobacter lactucae strain QL-1 possesses a superb DSF degradation ability and effectively attenuates Xcc virulence through QQ. However, the QQ mechanisms in strain QL-1 are still unknown. In the present study, whole-genome sequencing and comparative genomics analysis were conducted to identify the molecular mechanisms of QQ in strain QL-1. We found that the fadY gene of QL-1 is an ortholog of XccrpfB, a known DSF degradation gene, suggesting that strain QL-1 is capable of inactivating DSF by QQ enzymes. The results of site-directed mutagenesis indicated that fadY is required for strain QL-1 to degrade DSF. The determination of FadY activity in vitro revealed that the fatty acyl-CoA synthetase FadY had remarkable catalytic activity. Furthermore, the expression of fadY in transformed Xcc strain XC1 was investigated and shown to significantly attenuate bacterial pathogenicity on host plants, such as Chinese cabbage and radish. This is the first report demonstrating a DSF degradation enzyme from A. lactucae. Taken together, these findings shed light on the QQ mechanisms of A. lactucae strain QL-1, and provide useful enzymes and related genes for the biocontrol of infectious diseases caused by DSF-dependent bacterial pathogens.

The imbalance of gut microbiota is an important factor leading to inflammatory bowel disease (IBD). Diffusible signal factor (DSF) is a novel quorum-sensing signal that regulates bacterial growth, metabolism, pathogenicity, and host immune response. This study aimed to explore the therapeutic effect and underlying mechanisms of DSF in a zebrafish colitis model induced by sodium dextran sulfate (DSS). The results showed that intake of DSF can significantly improve intestinal symptoms in the zebrafish colitis model, including ameliorating the shortening of the intestine, reducing the increase in the goblet cell number, and restoring intestinal pathological damage. DSF inhibited the upregulation of inflammation-related genes and promoted the expression of claudin1 and occludin1 to protect the tightness of intestinal tissue. The gut microbiome analysis demonstrated that DSF treatment helped the gut microbiota of the zebrafish colitis model recover to normal at the phylum and genus levels, especially in terms of pathogenic bacteria; DSF treatment downregulated the relative abundance of Aeromonas hydrophila and Staphylococcus aureus, and it was confirmed in microbiological experiments that DSF could effectively inhibit the colonization and infection of these two pathogens in the intestine. This study suggests that DSF can alleviate colitis by inhibiting the proliferation of intestinal pathogens and inflammatory responses in the intestine. Therefore, DSF has the potential to become a dietary supplement that assists in the antibiotic and nutritional treatment of IBD.

V. cholerae continues to cause sporadic pandemics globally. Although cholera is a self-limiting acute disease, severe cases often require the use of antimicrobials in addition to rehydration therapy. Efforts to eradicate cholera have been hampered by the increased incidence of antimicrobial resistance and low vaccine coverage. The gut chemical environment, driven largely by the microbiota, has been shown to play important roles in modulating colonization by enteric pathogens. Pathogens utilize this chemical environment as cues to optimize virulence and survival. Understanding the underlying mechanisms of pathogen-microbiota interactions may therefore provide avenues to develop control measures targeting virulence and survival. Many of the mechanisms of pathogen-microbiota interactions have yet to be fully described. We demonstrate that V. cholerae integrates interspecies chemical signals (DSFs) produced by members of the microbiota to regulate the expression of important virulence factors. DSFs potently repress the virulence island master regulator (ToxT), effectively attenuating cholera toxin secretion. As other related long-chain fatty acids have been reported to impair ToxT function, and as DSF producers localize to the intestinal crypts and mucus layer, it is likely that V. cholerae employs these signals in a spatial-temporal manner to program the induction and repression of the energy-intensive virulence factors. Our work provides a framework for designing interventions to disrupt this virulence and survival program to control this pathogen.