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The distribution and transmission of antibiotic resistance genes (ARGs) in agricultural soils constitute a significant threat to food safety and human health. Natural quorum sensing inhibitors (QSIs), with advantages such as low plant toxicity and low application costs, present a potential approach for mitigating ARG contamination by targeting bacterial quorum sensing systems. This study explored the impacts and mechanisms of three natural QSIs (vanillin, catechin, and tannin) on the abundance of tetracycline resistance genes (TRGs) in both rhizosphere and non-rhizosphere soils. Results illustrated a notable reduction in TRG abundance across three natural QSI treatments, with catechin displaying the most pronounced effect in the rhizosphere soil. Furthermore, the application of natural QSIs had a significant influence on the bacterial community structure and population dynamics, particularly evident in the alterations induced by catechin on bacterial interactions within the soil ecosystem. Natural QSIs inhibited the production of N-acyl homoserine lactone (AHL) signaling molecules. The primary environmental factors driving changes in bacterial community were identified as pH and NO3--N content. Through mechanisms involving the modulations of AHL concentrations and soil environmental factors, natural QSIs were found to impact bacterial population, ultimately leading to a decrease in TRG abundance. Importantly, the application of natural QSIs did not exhibit adverse effects on plant phenotypic traits. These findings serve as a useful reference for implementing natural QSIs to effectively control soil ARG contamination.

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The broad-spectrum antibacterial capabilities of fatty acids (FAs) and their reduced propensity to promote resistance have rendered as a promising substitute for conventional antibiotics. The structural significance of fatty acid production with the other lipids is a major energy source, and signal transduction has drawn a great deal of research attention to these biomolecules. Saturated and monounsaturated fatty acids reduce virulence by preventing harmful opportunistic bacteria like Pseudomonas aeruginosa and Chromobacterium violaceum from activating their quorum sensing (QS) systems. In this finding, the fatty acids capric acid, caprylic acid, and monoelaidin were selected to evaluate their anti-QS activity against the C. violaceum and P. aeruginosa. At the minimum inhibitory concentration (MIC) and sub-MIC concentration of the three fatty acids, the virulence factor production of both the bacteria was quantified. The virulence factors like EPS, biofilm quantification and visualization, and motility assays were inhibited in the dose-dependent manner (MIC and sub-MIC) for both the organisms whereas this pattern was followed in the pyocyanin, pyoverdine, rhamnolipid, protease of P. aeruginosa and the violacein, and chitinase of C. violaceum. In all these biochemical assays, the capric acid could effectively reduce the production and further validated at gene expression level by RT-qPCR. The study on the gene expression for all these virulence factors reveals that the capric acid inhibited the growth of both the organisms in a higher fold than the caprylic and monoelaidin. The in silico approach of structural validation for the binding of ligands with the proteins in the QS circuit was studied by molecular docking in Schrodinger software. The Las I and Las R in P. aeruginosa and the CviR of C. violaceum protein structures were docked with the selected three fatty acids. The capric acid binds to the pocket with the highest binding score of all the proteins than the caprylic and monoelaidin fatty acids. Thus, capric acid proves to be the therapeutic biomolecule for the anti-QS activity of opportunistic bacteria.

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Microorganisms are the most common cause of food spoilage. Pseudomonas aeruginosa is a common foodborne pathogen that causes food spoilage and poses a serious threat to food safety. As a crucial target in antitoxicity strategies, the quorum sensing (QS) system shows promising potential for further development. The garlic extract diallyl disulfide exhibits inhibitory activity against the QS system of P. aeruginosa, with disulfide bonds serving as the active component. However, the biological activity of other symmetric disulfides has not been investigated in this capacity. The study synthesized 39 disulfide bond-containing analogs and evaluated their activity as quorum sensing inhibitors (QSIs). The results showed that p-hydroxyphenyl substitution can replace the allyl groups while maintaining strong biological activity. The virulence factors production was reduced by compound 2i, with the strongest inhibitory effect being observed on elastase production. Synergistic inhibition was observed in the presence of antibiotics like ciprofloxacin and tobramycin. 2i successfully inhibited P. aeruginosa infection in the Galleria mellonella larvae model. Primary mechanism studies using transcriptome, surface plasmon resonance and molecular docking suggested that 2i inhibits the QS system by targeting the LasR protein. Thus, compound 2i could be used in developing QSIs for the control of P. aeruginosa infections.

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AIMS: Aeromonas hydrophila, a Gram-negative bacterium, is ubiquitously found in many aquatic habitats causing septicemia in humans and fishes. Attributed to abuse or misuse of conventional antimicrobial drug usage, antimicrobial resistance is at an alarming rise. There is an available alternative strategy to bacterial resistance to antimicrobials which is inhibition of virulence and pathogenicity employing quorum sensing inhibitors (QSIs). Hence, actinomycin D's effectiveness against A. hydrophila SHAe 115 as a quorum sensing inhibitor (QSI) was investigated in decreasing virulence factors and preventing biofilm formation. METHODS AND RESULTS: Actinomycin D, belongs to the QSI combating Pseudomonas aeruginosa PAO1 originally isolated from an entophytic actinomycete (Streptomyces cyaneochromogenes RC1) in Areca catechu L. In the present work, further investigations were carried out to assess the effect of actinomycin D at subminimal inhibitory concentrations (sub-MICs), QS-regulated virulence factors and biofilm inhibition strategies. Intrinsic properties encompassing inhibition of the production of protease and hemolysin and subsequent activities on biofilm formation and eradication of mature biofilm were established along with weakened swimming and swarming motilities in A. hydrophila SHAe 115. In the Tenebrio molitor survival assay, actinomycin D effectively reduced the virulence and pathogenicity of A. hydrophila, resulting in elimination of mortality. However, the hydrolysate of actinomycin D, 2-hydroxy-4,6-dimethyl-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid (HDPD), had lost the QSI activity in A. hydrophila. CONCLUSIONS: Actinomycin D was proved as a viable QSI in lessning A. hydrophila's the virulence and pathogenicity as evident from our research findings.

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The quorum sensing (QS) system mediated by the abaI gene in Acinetobacter baumannii is crucial for various physiological and pathogenic processes. In this study, we constructed a stable markerless abaI knockout mutant (ΔabaI) strain using a pEXKm5-based allele replacement method to investigate the impact of abaI on A. baumannii. Proteomic analysis revealed significant alterations in protein expression between the wild type (WT) and ΔabaI mutant strains, particularly in proteins associated with membrane structure, antibiotic resistance, and virulence. Notably, the downregulation of key outer membrane proteins such as SurA, OmpA, OmpW, and BamA suggests potential vulnerabilities in outer membrane integrity, which correlate with structural abnormalities in the ΔabaI mutant strain, including irregular cell shapes and compromised membrane integrity, observed by scanning and transmission electron microscopy. Furthermore, diminished expression of regulatory proteins such as OmpR and GacA-GacS highlights the broader regulatory networks affected by abaI deletion. Functional assays revealed impaired biofilm formation and surface-associated motility in the mutant strain, indicative of altered colonization capabilities. Interestingly, the mutant showed a complex antibiotic susceptibility profile. While it demonstrated increased susceptibility to membrane-targeting antibiotics, its response to beta-lactams was more nuanced. Despite increased expression of metallo-beta-lactamase (MBL) superfamily proteins and DcaP-like protein, the mutant unexpectedly showed lower MICs for carbapenems (imipenem and meropenem) compared to the wild-type strain. This suggests that abaI deletion affects antibiotic susceptibility through multiple, potentially competing mechanisms. Further investigation is needed to fully elucidate the interplay between quorum sensing, antibiotic resistance genes, and overall antibiotic susceptibility in A. baumannii. Our findings underscore the multifaceted role of the abaI gene in modulating various cellular processes and highlight its significance in A. baumannii physiology, pathogenesis, and antibiotic resistance. Targeting the abaI QS system may offer novel therapeutic strategies for this clinically significant pathogen.

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How bacterial pathogens exploit host metabolism to promote immune tolerance and persist in infected hosts remains elusive. To achieve this, we show that Pseudomonas aeruginosa (PA), a recalcitrant pathogen, utilizes the quorum sensing (QS) signal 2'-aminoacetophenone (2-AA). Here, we unveil how 2-AA-driven immune tolerization causes distinct metabolic perturbations in murine macrophages' mitochondrial respiration and bioenergetics. We present evidence indicating that these effects stem from decreased pyruvate transport into mitochondria. This reduction is attributed to decreased expression of the mitochondrial pyruvate carrier (Mpc1), which is mediated by diminished expression and nuclear presence of its transcriptional regulator, estrogen-related nuclear receptor alpha (Esrra). Consequently, Esrra exhibits weakened binding to the Mpc1 promoter. This outcome arises from the impaired interaction between Esrra and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a). Ultimately, this cascade results in diminished pyruvate influx into mitochondria and, consequently reduced ATP production in tolerized murine and human macrophages. Exogenously added ATP in infected macrophages restores the transcript levels of Mpc1 and Esrra and enhances cytokine production and intracellular bacterial clearance. Consistent with the in vitro findings, murine infection studies corroborate the 2-AA-mediated long-lasting decrease in ATP and acetyl-CoA and its association with PA persistence, further supporting this QS signaling molecule as the culprit of the host bioenergetic alterations and PA persistence. These findings unveil 2-AA as a modulator of cellular immunometabolism and reveal an unprecedented mechanism of host tolerance to infection involving the Ppargc1a/Esrra axis in its influence on Mpc1/OXPHOS-dependent energy production and PA clearance. These paradigmatic findings pave the way for developing treatments to bolster host resilience to pathogen-induced damage. Given that QS is a common characteristic of prokaryotes, it is likely that 2-AA-like molecules with similar functions may be present in other pathogens.

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The contagious respiratory pathogen, Avibacterium paragallinarum, contributes to infectious coryza in poultry. However, commercial vaccines have not shown perfect protection against infectious coryza. To search for an alternative approach, this research aimed to investigate whether the quorum-sensing system of pathogens plays a crucial role in their survival and pathogenicity. The LuxS/AI-2 quorum-sensing system in many Gram-negative and Gram-positive bacteria senses environmental changes to regulate physiological traits and virulent properties, and the role of the luxS gene in Av. paragallinarum remains unclear. To investigate the effect of the luxS gene in the quorum-sensing system of Av. paragallinarum, we constructed a luxS mutant. Bioluminescence analysis indicated that the luxS gene plays a vital role in the LuxS/AI-2 quorum-sensing system. The analysis of the LuxS/AI-2 system-related genes showed the level of pfs mRNA to be significantly increased in the mutant strain; however, lsrR, lsrK, and lsrB mRNA levels were not significantly different compared with the wild type. The ability of the luxS mutant strain to invade HD11 and DF-1 cells was significantly decreased compared with the wild-type strain. In addition, all chickens challenged with various doses of the luxS mutant strain developed infections and symptoms, and those challenged with the lowest dose exhibited only minor differences compared to chickens challenged with the wild-type strain. Thus, the deletion of the luxS gene reduces the invasion, but the luxS gene does not play an essential role in the pathogenesis of A. paragallinarum.

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In an environment, microbes often work in communities to achieve most of their essential functions, including the production of essential nutrients. Microbial biofilms are communities of microbes that attach to a nonliving or living surface by embedding themselves into a self-secreted matrix of extracellular polymeric substances. These communities work together to enhance their colonization of surfaces, produce essential nutrients, and achieve their essential functions for growth and survival. They often consist of diverse microbes including bacteria, viruses, and fungi. Biofilms play a critical role in influencing plant phenotypes and human microbial infections. Understanding how these biofilms impact plant health, human health, and the environment is important for analyzing genotype-phenotype-driven rule-of-life functions. Such fundamental knowledge can be used to precisely control the growth of biofilms on a given surface. Metagenomics is a powerful tool for analyzing biofilm genomes through function-based gene and protein sequence identification (functional metagenomics) and sequence-based function identification (sequence metagenomics). Metagenomic sequencing enables a comprehensive sampling of all genes in all organisms present within a biofilm sample. However, the complexity of biofilm metagenomic study warrants the increasing need to follow the Findability, Accessibility, Interoperability, and Reusable (FAIR) Guiding Principles for scientific data management. This will ensure that scientific findings can be more easily validated by the research community. This study proposes a dockerized, self-learning bioinformatics workflow to increase the community adoption of metagenomics toolkits in a metagenomics and meta-transcriptomics investigation. Our biofilm metagenomics workflow self-learning module includes integrated learning resources with an interactive dockerized workflow. This module will allow learners to analyze resources that are beneficial for aggregating knowledge about biofilm marker genes, proteins, and metabolic pathways as they define the composition of specific microbial communities. Cloud and dockerized technology can allow novice learners-even those with minimal knowledge in computer science-to use complicated bioinformatics tools. Our cloud-based, dockerized workflow splits biofilm microbiome metagenomics analyses into four easy-to-follow submodules. A variety of tools are built into each submodule. As students navigate these submodules, they learn about each tool used to accomplish the task. The downstream analysis is conducted using processed data obtained from online resources or raw data processed via Nextflow pipelines. This analysis takes place within Vertex AI's Jupyter notebook instance with R and Python kernels. Subsequently, results are stored and visualized in Google Cloud storage buckets, alleviating the computational burden on local resources. The result is a comprehensive tutorial that guides bioinformaticians of any skill level through the entire workflow. It enables them to comprehend and implement the necessary processes involved in this integrated workflow from start to finish. This manuscript describes the development of a resource module that is part of a learning platform named "NIGMS Sandbox for Cloud-based Learning" https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

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The opportunistic pathogen Pseudomonas aeruginosa has complex quorum sensing (QS) circuitry, which involves two acylhomoserine lactone (AHL) systems, the LasI AHL synthase and LasR AHL-dependent transcriptional activator system and the RhlI AHL synthase-RhlR AHL-responsive transcriptional activator. There is also a quinoline signaling system [the Pseudomonas quinolone signal (PQS) system]. Although there is a core set of genes regulated by the AHL circuits, there is strain-to-strain variation in the non-core QS regulon. A size reduction of the QS regulon occurs in laboratory evolution experiments with the model strain PAO1. We used transcriptomics to test the hypothesis that reductive evolution in the PAO1 QS regulon can in large part be explained by a null mutation in pqsR, the gene encoding the transcriptional activator of the pqs operon. We found that PqsR had very little influence on the AHL QS regulon. This was a surprising finding because the last gene in the PqsR-dependent pqs operon, pqsE, codes for a protein, which physically interacts with RhlR, and this interaction is required for RhlR-dependent activation of some genes. We used comparative transcriptomics to examine the influence of a pqsE mutation on the QS regulon and identified only three transcripts, which were strictly dependent on PqsE. By using reporter constructs, we showed that the PqsE influence on other genes was dependent on experimental conditions and we have gained some insight about those conditions. This work adds to our understanding of the plasticity of the P. aeruginosa QS regulon and to the role PqsE plays in RhlR-dependent gene activation.IMPORTANCEOver many generations of growth in certain conditions, Pseudomonas aeruginosa undergoes a large reductive evolution in the number of genes activated by quorum sensing. Here, we rule out one plausible route of the reductive evolution: that a mutation in a transcriptional activator PqsR or the PqsR activation of pqsE, which codes for a chaperone for the quorum sensing signal-responsive transcription factor RhlR, explains the finding. We further provide information about the influence of PqsR and PqsE on quorum sensing in P. aeruginosa.

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The SorC family is a large group of bacterial transcription regulators involved in controlling carbohydrate catabolism and quorum sensing. SorC proteins consist of a conserved C-terminal effector-binding domain and an N-terminal DNA-binding domain, whose type divides the family into two subfamilies: SorC/DeoR and SorC/CggR. Proteins of the SorC/CggR subfamily are known to regulate the key node of glycolysis-triose phosphate interconversion. On the other hand, SorC/DeoR proteins are involved in a variety of peripheral carbohydrate catabolic pathways and quorum sensing functions, including virulence. Despite the abundance and importance of this family, SorC proteins seem to be on the periphery of scientific interest, which might be caused by the fragmentary information about its representatives. This review aims to compile the existing knowledge and provide material to inspire future questions about the SorC protein family.

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To construct a derivative of the avirulent Pseudomonas aeruginosa ATCC 9027 that produces high levels of di-rhamnolipid, that has better physico-chemical characteristics for biotechnological applications than mono-rhamnolipid, which is the sole type produced by ATCC 9027. We used plasmids expressing the rhlC gene, which encodes for rhamnosyl transferase II that transforms mono- to di-rhamnolipids under different promoters and in combination with the gene coding for the RhlR quorum sensing regulator, or the mono-rhamnolipid biosynthetic rhlAB operon. The plasmids tested carrying the rhlC gene under the lac promoter were plasmid prhlC and prhlRC, while prhlAB-R-C expressed this gene from the rhlA promoter, forming part of the artificially constructed rhlAB-R-C operon. We measured rhamnolipds concentrations using the orcinol method and determined the proportion of mono-rhamnolipids and di-rhamnolipids by UPLC/MS/MS. We found that the expression of rhlC in P. aeruginosa ATCC 9027 caused the production of di-rhamnolipids and that the derivative carrying plasmid prhlAB-R-C gives the best results considering total rhamnolipids and a higher proportion of di-rhamnolipids. A P. aeruginosa ATCC 9027 derivative with increased di-rhamnolipids production was developed by expressing plasmid prhlAB-R-C, that produces similar rhamnolipids levels as PAO1 type-strain and presented a higher proportion of di-rhamnolipids than this type-strain.

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Quorum sensing (QS) signals widely exist in bacteria to control biological functions in response to populations of cells. Burkholderia cenocepacia, an important opportunistic pathogen in patients with cystic fibrosis (CF), is commonly found in the environment and mostly utilizes the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) QS systems to control biological functions. Our previous study illuminated the detailed mechanism by which B.cenocepacia integrates BDSF and cyclic diguanosine monophosphate (c-di-GMP) signals to control virulence. Here, we employed Tn5 transposon mutagenesis to identify genes related to the BDSF QS system. One of the most significantly attenuated mutants had an insertion in the mntH gene. Here, we showed that MntH (Bcam0836), a manganese transport protein, controls QS-regulated phenotypes, including motility, biofilm formation and virulence. We also found that. BDSF production was attenuated at both the gene and signaling levels in the Bcam0836 mutant, and that Bcam0836 influenced the expression of some genes regulated by the BDSF receptor RpfR and the downstream regulator GtrR. These results show that the manganese transport protein. MntH modulates a subset of genes and functions regulated by the QS system in B. cenocepacia.

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Antibiotic resistance is one of the most important concerns in global health today. Thus, a growing number of different infections are becoming harder to treat with conventional drugs and this is aggravated by the fact that fewer new antibiotics are being developed. In this context, strategies based on blocking or attenuating virulence pathways could position as very interesting therapeutic approaches since they do not focus on bacteria eradication, which should reduce the selective pressure exerted on the pathogen. This virulence depletion can be achieved by inhibiting the conserved quorum sensing (QS) system, a mechanism that enables bacteria to communicate one another in a density dependent manner. QS regulates gene expression leading to the activation of some important processes such as virulence and biofilm formation among others. Therefore, this review points out the approaches reported so far for disrupting different steps of the QS system of the multiresistant pathogen Pseudomonas aeruginosa. With this aim, the authors describe different types of molecules (enzymes, natural and synthetic small molecules, antibodies…) already identified as P. aeruginosa quorum quenchers (QQs) or QS inhibitors (QSIs) grouped according to the QS circuit that they block (Las, Rhl, Pqs and some examples from the controversial pathway Iqs). The importance of the discovery of new QSIs and QQs is expected to help on reducing antibiotic doses or at least to act as adjuvants to increase antibiotic treatment effect. Moreover, this article also highlights the advantages and possible drawbacks of each strategy and it also summarizes future perspectives in the field.

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This study aims to investigate the drug resistance, regulation mechanism of quorum sensing system, expression of related virulence genes, and epidemiological characteristics of carbapenem-resistant Pseudomonas aeruginosa (CRPA).In this study, Polymerase chain reaction amplification was performed to evaluate carbapenemase genes, OprD2 gene, quorum sensing system, and related virulence genes. Bacterial genotypes were analyzed using multilocus sequence typing and evolutionary analysis was conducted based on the goeBURST algorithm. The results demonstrated that a total of 47 CRPA strains were collected in this study, primarily from respiratory specimens in the ICU. Drug sensitivity results showed that the resistance rates of the 47 CRPA strains were highest for imipenem (97.87 %). The loss of OprD2 may be the main factor contributing to carbapenem resistance in our hospital's CRPA strains.All isolates tested positive for the quorum sensing system genes lasI and rhlI/R, and the virulence gene lasB was detected in all isolates, while the algD gene was detected in 19.15 % of the isolates. Among the 47 strains, 6 were untypeable, and the 41 strains with 28 different sequence types were clustered into three clonal complexes (BG1, BG2, and BG3).In conclusion, the CRPA isolates from our hospital exhibit high genetic diversity, with the deletion of the OprD2 gene possibly being the primary determinant of carbapenem resistance in Pseudomonas aeruginosa.Moreover, Las and RhI systems play a key role in quorum sensing signal system. Further research and development of drugs targeting quorum sensing signaling system may provide valuable guidance for the treatment of CRPA.

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The luxS/AI-2 quorum sensing (QS) system of Streptococcus thermophilus regulates strain acid tolerance, yet its impact on milk fermentation remains unclear. This study aimed to elucidate the mechanism of luxS and pfs gene overexpression in the luxS/AI-2 system of S. thermophilus ABT-T on fermented milk quality using metabolomics. Results showed that pfs gene overexpression had a greater impact on milk quality than the wild-type strain or luxS gene overexpression strain. Overexpression of the pfs gene significantly enhanced AI-2 secretion, reducing fermented milk pH, increasing acidity, improving fermented milk protein hydrolysis, and altering texture and water-holding capacity. Nineteen volatile flavor compounds were identified, with decreased ketone compounds due to the pfs gene overexpression. KEGG analysis suggested significant alterations in amino acid metabolism pathways due to the pfs gene overexpression. This study provides insights into the role of QS in fermented foods.

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Introduction: Pseudomonas aeruginosa is a ubiquitous pathogen that causes various infectious diseases through the regulation of quorum sensing (QS). The strategy of interfering with the QS systems of P. aeruginosa, coupled with a reduction in the dosage of conventional antibiotics, presents a potential solution to treating infection and mitigating antibiotic resistance. In this study, seven cinnamoyl hydroxamates were synthesized to evaluate their inhibitory effects on QS of P. aeruginosa. Among these cinnamic acid derivatives, we found cinnamoyl hydroxamic acid (CHA) and 3-methoxy-cinnamoyl hydroxamic acid (MCHA) were the two most effective candidates. Furtherly, the effect of CHA and MCHA on the production of virulence factors and biofilm of P. aeruginosa were evaluated. Ultimately, our study may offer promising potential for treating P. aeruginosa infections and reducing its virulence. Methods: The disc diffusion test were conducted to evaluate inhibitory effects on QS of seven cinnamoyl hydroxamates. The influence of CHA and MCHA on the production of virulence and flagellar motility of P. aeruginosa was furtherly explored. Scanning electron microscopy (SEM) experiment were conducted to evaluate the suppression of CHA and MCHA on the formed biofilm of P. aeruginosa. RT-qPCR was used to detect rhlI, lasA, lasB, rhlA, rhlB, and oprL genes in P. aeruginosa. In silico docking study was performed to explore the molecular mechanism of CHA and MCHA. The synergistic effects of CHA with gentamicin were detected on biofilm cell dispersal. Result: After treatment of CHA or MCHA, the production of multiple virulence factors, including pyocyanin, proteases, rhamnolipid, and siderophore, and swimming and swarming motilities in P. aeruginosa were inhibited significantly. And our results showed CHA and MCHA could eliminate the formed biofilm of P. aeruginosa. RT-qPCR revealed that CHA and MCHA inhibited the expression of QS related genes in P. aeruginosa. Molecular docking indicated that CHA and MCHA primarily inhibited the RhlI/R system in P. aeruginosa by competing with the cognate signaling molecule C4-HSL.Additionally, CHA exhibited potent synergistic effects with gentamicin on biofilm cell dispersal. Discussion: P. aeruginosa is one of the most clinically and epidemiologically important bacteria and a primary cause of catheter-related urinary tract infections and ventilator-associated pneumonia. This study aims to explore whether cinnamoyl hydroxamates have inhibitory effects on QS. And our results indicate that CHA and MCHA, as two novel QSIs, offer promising potential for treating P. aeruginosa infections and reducing its virulence.

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Orthopedic implant-related bacterial infections and resultant antibiotic-resistant biofilms hinder implant-tissue integration and failure. Biofilm quorum sensing (QS) communication determines the pathogen colonization success. However, it remains unclear how implant modifications and host cells are influenced by, or influence, QS. High aspect ratio nanotopographies have shown to reduce biofilm formation of Pseudomonas aeruginosa, a sepsis causing pathogen with well-defined QS molecules. Producing such nanotopographies in relevant orthopedic materials (i.e., titanium) allows for probing QS using mass spectrometry-based metabolomics. However, nanotopographies can reduce host cell adhesion and regeneration. Therefore, we developed a polymer (poly(ethyl acrylate), PEA) coating that organizes extracellular matrix proteins, promoting bioactivity to host cells such as human mesenchymal stromal cells (hMSCs), maintaining biofilm reduction. This allowed us to investigate how hMSCs, after winning the race for the surface against pathogenic cells, interact with the biofilm. Our approach revealed that nanotopographies reduced major virulence pathways, such as LasR. The enhanced hMSCs support provided by the coated nanotopographies was shown to suppress virulence pathways and biofilm formation. Finally, we selected bioactive metabolites and demonstrated that these could be used as adjuncts to the nanostructured surfaces to reduce biofilm formation and enhance hMSC activity. These surfaces make excellent models to study hMSC-pathogen interactions and could be envisaged for use in novel orthopedic implants.

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Increased prevalence of multidrug-resistant bacterial infections has sparked interest in alternative antimicrobials, including bacteriophages (phages). Limited understanding of the phage infection process hampers our ability to utilize phages to their full therapeutic potential. To understand phage infection dynamics, we performed proteomics on Enterococcus faecalis infected with the phage VPE25. We discovered that numerous uncharacterized phage proteins are produced during phage infection of E. faecalis. Additionally, we identified hundreds of changes in bacterial protein abundances during infection. One such protein, enterococcal gelatinase (GelE), an fsr quorum-sensing-regulated protease involved in biofilm formation and virulence, was reduced during VPE25 infection. Plaque assays showed that mutation of either the quorum-sensing regulator fsrA or gelE resulted in plaques with a "halo" morphology and significantly larger diameters, suggesting decreased protection from phage infection. GelE-associated protection during phage infection is dependent on the putative murein hydrolase regulator LrgA and antiholin-like protein LrgB, whose expression have been shown to be regulated by GelE. Our work may be leveraged in the development of phage therapies that can modulate the production of GelE thereby altering biofilm formation and decreasing E. faecalis virulence.

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Quorum sensing (QS) plays an important role in the social behavior of microbial communities. Anaerobic digestion (AD) is a biological process using anaerobic microorganisms to degrade organic macromolecules into small molecules for biogas and biofertilizer production. In AD, the QS signaling molecule N-acyl homoserine lactones (AHLs) induces bacterial metabolism, improving AD process efficiency. However, there are fewer systematic reports about QS regulation of microbial behavior in AD. In this report, the effects of signaling molecules on extracellular polymer secretion, biofilm formation, granulation of granular sludge and bacterial metabolism in AD were investigated in detail. At present, the regulation behavior of QS on AD is a group phenomenon, and there are few in-depth studies on the regulation pathway. Therefore, we conducted an in-depth analysis of the pure culture system, granular sludge and reactor in the AD. Then we pointed out that the future application potential of QS in the AD may be combined with quorum quenching (QQ) and omics technology, which is of great significance for the future application of AD.

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The accessory gene regulatory (Agr) system is required for virulence factor gene expression and pathogenesis of Staphylococcus aureus. The Agr system is activated in response to the accumulation of a cyclic autoinducing peptide (AIP), which is matured and secreted by the bacterium. The precursor of AIP, AgrD, consists of the AIP flanked by an N-terminal [Formula: see text]-helical Leader and a charged C-terminal tail. AgrD is matured to AIP by the action of two proteases, AgrB and MroQ. AgrB cleaves the C-terminal tail and promotes the formation of a thiolactone ring, whereas MroQ cleaves the N-terminal Leader in a manner that depends on the four-amino acid linker immediately following a conserved IG helix breaker motif. However, the attributes of AgrD that dictate the sequence of events in peptide maturation are not fully defined. Here, we used engineered AgrD peptide intermediates to ascertain the sufficiency of MroQ for N-terminal peptide cleavage, peptide export, and generation of mature AIP. We found that MroQ promotes the removal of the N-terminal Leader peptide from both linear and cyclic peptide intermediates, while peptide cyclization remained essential for signaling. The expression of the Leader peptide in isolation was sufficient for MroQ-dependent cleavage proximal to the four-amino-acid linker. In addition, active site mutations within AgrB destabilized full-length AgrD and thiolactone-containing intermediates and prevented the release of the Leader peptide. Altogether, our data support a tandem peptide maturation event involving both MroQ and AgrB that appears to couple protease activity and export of bioactive AIP.IMPORTANCEThe accessory gene regulatory (Agr) system is important for S. aureus pathogenesis. Activation of the Agr system requires recognition of a cyclic peptide pheromone, which must be fully matured to exert its biological activity. The complete events in cyclic peptide maturation and export from the bacterial cell remain to be fully defined. We and others recently discovered that the membrane peptidase MroQ is required for pheromone maturation. This study builds off the identification of MroQ and considers the attributes of the pheromone pro-peptide that are required for MroQ-mediated processing as well as uncovers features important for peptide stability and export. Overall, the findings in this study have implications for understanding bacterial pheromone maturation and virulence.