Synergistic toxic effects of high-strength ammonia and ZnO nanoparticles on biological nitrogen removal systems and role of exogenous C10-HSL regulation.

The inhibitory effects of zinc oxide nanoparticles (ZnO NPs) and impacts of N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) on biological nitrogen removal (BNR) performance have been well-investigated. However, the effects of ammonia nitrogen (NH4+-N) concentrations on NP toxicity and AHL regulation have seldom been addressed yet. This study consulted on the impacts of ZnO NPs on BNR systems when high NH4+-N concentration was available. The synergistic toxic effects of high-strength NH4+-N (200 mg/L) and ZnO NPs resulted in decreased ammonia oxidation rates and dropped the nitrogen removal efficiencies by 17.5% ± 0.2%. The increased extracellular polymeric substances (EPS) production was observed in response to the high NH4+-N and ZnO NP stress, which indicated the defense mechanism against the toxic effects in the BNR systems was stimulated. Furthermore, the regulatory effects of exogenous N-decanoyl-homoserine lactone (C10-HSL)-mediated QS system on NP-stressed BNR systems were revealed to improve the BNR performance under different NH4+-N concentrations. The C10-HSL regulated the intracellular reactive oxygen species levels, denitrification functional enzyme activities, and antioxidant enzyme activities, respectively. This probably synergistically enhanced the defense mechanism against NP toxicity. However, compared to the low NH4+-N concentration of 60 mg/L, the efficacy of C10-HSL was inhibited at high NH4+-N levels of 200 mg/L. The findings provided the significant application potential of QS system for BNR when facing toxic compound shock threats.

Free-swimming bacteria transcriptionally respond to shear flow.

Surface-attached cells can sense and respond to shear flow, but planktonic (free-swimming) cells are typically assumed to be oblivious to any flow that carries them. Here, we find that planktonic bacteria can transcriptionally respond to flow, inducing expression changes that are beneficial in flow. Specifically, we use microfluidic experiments and quantitative modeling to show that in the presence of flow, planktonic Pseudomonas aeruginosa induce shear rate-dependent genes that promote growth in low-oxygen environments. Untangling this mechanism revealed that in flow, motile P. aeruginosa spatially redistribute, leading to cell density changes that activate quorum sensing, which in turn enhances the oxygen uptake rate. In diffusion-limited environments, including those commonly encountered by bacteria, flow-induced cell density gradients also independently generate oxygen gradients that alter gene expression. Mutants deficient in this flow-responsive mechanism exhibit decreased fitness in flow, suggesting that this dynamic coupling of biological and mechanical processes can be physiologically significant.

Cinnamoyl lipids as novel signaling molecules modulate the physiological metabolism of cross-phylum microorganisms.

Signaling systems of microorganisms are responsible for regulating the physiological and metabolic processes and also play vital roles in the communications of cells. Identifying signaling molecules mediating the cross-talks is challenging yet highly desirable for comprehending the microbial interactions. Here, we demonstrate that a pathogenic Gram-negative Chromobacterium violaceum exerts significant influence on the morphological differentiation and secondary metabolism of Gram-positive Streptomyces. The physiological metabolisms are directly modulated by three novel cinnamoyl lipids (CVCL1, 2, and 3) from C. violaceum CV12472, whose biosynthesis is under the control of N-acylhomoserine lactone signaling system. Furthermore, a receptor of CVCLs in Streptomyces ansochromogenes 7100 is determined to be SabR1, the cognate receptor of γ-butenolide signaling molecules. This study reveals an unprecedented mode of microbial interactions, and the quorum sensing signaling systems in these two groups of bacteria can be bridged via CVCLs, suggesting that CVCLs can modulate the physiological metabolism of cross-phylum microorganisms.

[Advances in microbial whole-cell sensors for the detection of aromatic compounds].

The inherent stability and recalcitrance of benzene ring structures render aromatic compounds a major ecological concern and a substantial risk to human health. Hence, developing a facile and efficacious detection technique for aromatic compounds is essential. As our comprehension of aromatic compound characteristics deepens, microbial cell-based biosensors have emerged as increasingly popular tools in the detection of aromatic compounds. This article introduces the operational principles of microbial whole-cell biosensors and elucidates the construction techniques and applications of electroactive biofilm-based microbial whole-cell sensors, transcription factor-based microbial whole-cell sensors, and degradation gene promoter-dependent microbial whole-cell sensors in the detection of aromatic compounds. In addition, we review the methodologies for improving the performance of microbial whole-cell sensors based on surface display, logic gate construction, genetic circuit modification, and quorum sensing signal amplification.

Reducing tetracycline resistance genes in wheat soil using natural quorum sensing inhibitors: A new approach for mitigating antibiotic resistance gene contamination.

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.

Actinomycin D reduces virulence factors and biofilms against Aeromonas hydrophila.

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 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 lessening A. hydrophila's the virulence and pathogenicity, as evident from our research findings.

Contribution of the Sensor Histidine Kinases PhcS and VsrA to the Quorum Sensing of Ralstonia pseudosolanacearum Strain OE1-1.

The soilborne Gram-negative phytopathogenic beta-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate (3-OH MAME) as the quorum sensing (QS) signal by the methyltransferase PhcB and senses the chemical, activating the LysR family transcriptional regulator PhcA, which regulates the QS-dependent genes responsible for QS-dependent phenotypes including virulence. The sensor histidine kinases PhcS and VsrA are reportedly involved in the regulation of QS-dependent genes. To elucidate the function of PhcS and VsrA in the active QS, we generated the phcS-deletion and vsrA-deletion mutants, which exhibited weak changes to their QS-dependent phenotypes including virulence. The phcS and vsrA-deletion mutant (ΔphcS/vsrA) had significant changes in its QS-dependent phenotypes and was nonvirulent, similar to the phcA-deletion mutant. The mutant (PhcS-H230Q) with a substitution of histidine to glutamine at amino acid position 230 in PhcS but not the mutant (VsrA-H256Q) with a substitution of histidine to glutamine at amino acid position 256 in VsrA exhibited significant changes in QS-dependent phenotypes and lost virulence. The transcriptome analysis with RNA-sequencing revealed significant alterations to the expression of QS-dependent genes in the ΔphcS/vsrA and PhcS-H230Q but not VsrA-H256Q, similar to the phcA-deletion mutant. The exogenous 3-OH MAME application led to a significantly enhanced QS-inducible major exopolysaccharide EPS I production of the strain OE1-1 and phcB-deletion mutant but not ΔphcS/vsrA and PhcS-H230Q. Collectively, results of the present genetic study suggested that PhcS contributes to QS along with VsrA and that histidine at amino acid position 230 of PhcS is required for 3-OH MAME sensing, thereby influencing QS-dependent phenotypes including virulence of the strain OE1-1. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Hybrids of 3-Hydroxypyridin-4(1H)-ones and Long-Chain 4-Aminoquinolines as Potent Biofilm Inhibitors of Pseudomonas aeruginosa Potentiate Tobramycin and Polymyxin B Activity.

The biofilm formation of Pseudomonas aeruginosa involves multiple complex regulatory pathways; thus, blocking a single pathway is unlikely to achieve the desired antibiofilm efficacy. Herein, a series of hybrids of 3-hydroxypyridin-4(1H)-ones and long-chain 4-aminoquinolines were synthesized as biofilm inhibitors against P. aeruginosa based on a multipathway antibiofilm strategy. Comprehensive structure-activity relationship studies identified compound 30b as the most valuable antagonist, which significantly inhibited P. aeruginosa biofilm formation (IC50 = 5.8 µM) and various virulence phenotypes. Mechanistic studies revealed that 30b not only targets the three quorum sensing systems but also strongly induces iron deficiency signals in P. aeruginosa. Furthermore, 30b demonstrated a favorable in vitro and in vivo safety profile. Moreover, 30b specifically enhanced the antibacterial activity of tobramycin and polymyxin B in in vitro and in vivo combination therapy. Overall, these results highlight the potential of 30b as a novel anti-infective candidate for treating P. aeruginosa infections.

The role of male hormones in bacterial infections: enhancing Staphylococcus aureus virulence through testosterone-induced Agr activation.

Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.

Effects of AI-2 quorum sensing related luxS gene on Streptococcus suis formatting monosaccharide metabolism-dependent biofilm.

Biofilm is the primary cause of persistent infections caused by Streptococcus suis (S. suis). Metabolism and AI-2 quorum sensing are intricately linked to S. suis biofilm formation. Although the role of the AI-2 quorum sensing luxS gene in S. suis biofilm has been reported, its specific regulatory mechanism remains unclear. This study explored the differences in biofilm formation and monosaccharide metabolism among the wild type (WT), luxS mutant (ΔluxS) and complement strain (CΔluxS), and Galleria mellonella larvae were used to access the effect of luxS gene deletion on the virulence of S. suis in different monosaccharide medias. The results indicated that deletion of the luxS gene further compromised the monosaccharide metabolism of S. suis, impacting its growth in media with fructose, galactose, rhamnose, and mannose as the sole carbon sources. However, no significant impact was observed in media with glucose and N-acetylglucosamine. This deletion also weakened EPS synthesis, thereby diminishing the biofilm formation capacity of S. suis. Additionally, the downregulation of adhesion gene expression due to luxS gene deletion was found to be independent of the monosaccharide medias of S. suis.

Pseudomonas aeruginosa Mediates Host Necroptosis through Rhl-Pqs Quorum Sensing Interaction.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that can cause serious infections in immunocompromised patients. Quorum sensing (QS), a communication system evolved by P. aeruginosa to survey its density, is well acknowledged to be involved in various activities during bacterial infection. Recent studies have revealed the link between P. aeruginosa QS and host innate immune response. Previous evidence suggests that programmed cell death exists in response to P. aeruginosa infection. However, it remains unclear whether QS plays a role in the host programmed cell death process during the infection. In this study, we found that the deficiency of one of QS subsystems, rhl, markedly increased mouse bone marrow macrophage cell death induced by P. aeruginosa, which was accompanied by elevated phosphorylation of RIPK3 and MLKL. This highly increased necroptosis activation was caused by the upregulation of another QS subsystem, pqs, because the deletion of pqs in rhl-deficient P. aeruginosa abolished macrophage necroptosis in vitro and in vivo. In sum, our data highlight the cross-talk between P. aeruginosa QS and host necroptosis, which is executed through the rhl-pqs axis.

Cyclic AMP is a global virulence regulator governing inter and intrabacterial signalling in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic nosocomial pathogen with high morbidity and mortality rates. Current treatment options for this pathogen are limited due to its increasing resistance to last-resort antibiotics. Despite A. baumannii's leading position in the World Health Organisations priority pathogens list, little is known about its virulence regulation. Through a high-throughput screening approach to identify novel biofilm regulators, we identified a previously uncharacterised predicted adenylate cyclase (AC), CavA, as a central regulator of this phenotype. cAMP is a crucial mediator of various aspects of bacterial physiology in other species but information about its role in A. baumannii is limited. We confirm that CavA AC is functional and synthesizes cAMP in A. baumannii. Using dRNA-seq, we verify that CavA is a negative biofilm formation regulator affecting Csu pili and exopolysaccharide production. We demonstrate for the first time that in A. baumannii, cAMP is atop of a hierarchical signalling cascade controlling inter- and intrabacterial signalling by modulating quorum sensing and cyclic di-GMP systems, ultimately governing virulence in vivo and adaptive antibiotic resistance. In contrast to the well-established paradigm in other bacteria where cAMP and cyclic di-GMP levels are inversely regulated, we uncover that the levels of these second messengers are directly proportional in A. baumannii. Overall, this study uncovers the central role of CavA and cAMP in the pathogenic success of A. baumannii and highlights this signalling cascade as a high potential target for novel therapeutic development.

The Comparative Characterization of a Hypervirulent Acinetobacter baumannii Bacteremia Clinical Isolate Reveals a Novel Mechanism of Pathogenesis.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen with exquisite survival capabilities under various environmental conditions and displays widespread resistance to common antibiotics. A. baumannii is a leading cause of nosocomial infections that result in high morbidity and mortality rates. Accordingly, when multidrug resistance rates surpass threshold levels, the percentage of A. baumannii clinical isolates surges. Research into A. baumannii has increased in the past decade, and multiple mechanisms of pathogenesis have been identified, including mechanisms underlying biofilm development, quorum sensing, exotoxin production, secretion system utilization, and more. To date, the two gold-standard strains used to investigate different aspects of A. baumannii pathogenesis include ATCC 17978 and ATCC 19606. Here, we report a comparative characterization study of three additional A. baumannii clinical isolates obtained from different infection types and derived from different anatomical regions of infected patients. The comparison of three clinical isolates in addition to the ATCC strains revealed that the hypervirulent bacteremia clinical isolate, known as HUMC1, employs a completely different mechanism of pathogenesis when compared to all its counterparts. In stark contrast to the other genetic variants, the hypervirulent HUMC1 isolate does not form biofilms, is antibiotic-susceptible, and has the capacity to reach higher levels of quorum compared to the other clinically relevant strains. Our data also reveal that HUMC1 does not shed endotoxin into the extracellular milieu, rather secretes the evolutionarily conserved, host-mimicking, Zonula occludens toxin (Zot). Taken together, our hypothesis that HUMC1 cells have the ability to reach higher levels of quorum and lack biofilm production and endotoxin shedding, accompanied by the substantial elaboration of Zot, suggests a novel mechanism of pathogenesis that appears to afford the hypervirulent pathogen with stealth-like capabilities when disseminating through the circulatory system in a state of bacteremia.

Heterologous expression of the Oenococcus oeni two-component signal transduction response regulator in the Lactiplantibacillus plantarum WCFS1 strain enhances acid stress tolerance.

BACKGROUND: Oenococcus oeni is a commercial wine-fermenting bacterial strain, owing to its high efficiency of malolactic fermentation and stress tolerance. The present study explored the function of key genes in O. oeni to enhance stress resistance by heterologous expression of these genes in another species. RESULTS: The orf00404 gene that encodes a two-component signal transduction response regulator in O. oeni was heterologously expressed in Lactiplantibacillus plantarum WCFS1. The expression of orf00404 significantly enhanced the growth rate of the recombinant strain under acid stress. At 60 h, 72 h, and 108 h of culture at pH 4.0, the recombinant strain had 1562, 641, and 748 differentially expressed genes compared to the control strain, respectively. At all three time points, 20 genes were upregulated in the recombinant strain, including the lamA-D operon-coding genes of the quorum-sensing two component signal transduction system and the spx5 RNA polymerase-binding protein coding gene, which may help adaptation to acid stress. In addition, 47 genes were downregulated in the recombinant strain at all three time points, including the hsp1 heat shock protein-coding gene, the trxA1 thioredoxin-coding gene, and the dinP, mutY, umuC, and uvrB DNA damage repair-related protein-coding genes, potentially indicating that the recombinant strain was less susceptible to stress and had less DNA damage than the control strain in acid stress conditions. The recombinant strain had higher membrane fluidity, permeability, and integrity at an early stage of logarithmic growth (72 h), suggesting that it had a more complete and active cell membrane state at this stage. The intracellular ATP content was significantly reduced in the recombinant strain at the beginning of logarithmic growth (60 h), implying that the recombinant strain consumed more energy at this stage to resist acid stress and growth. CONCLUSIONS: These results indicated that the recombinant strain enhances acid stress tolerance by regulating a gene expression pattern, increasing ATP consumption, and enhancing cell membrane fluidity, membrane permeability, and membrane integrity at specific growth stages. Thus, the recombinant strain may have potential application in the microbial biotechnology industry.

Engineering quorum sensing-based genetic circuits enhances growth and productivity robustness of industrial E. coli at low pH.

BACKGROUND: Microbial organisms hold significant potential for converting renewable substrates into valuable chemicals. Low pH fermentation in industrial settings offers key advantages, including reduced neutralizer usage and decreased wastewater generation, particularly in the production of amino acids and organic acids. Engineering acid-tolerant strains represents a viable strategy to enhance productivity in acidic environments. Synthetic biology provides dynamic regulatory tools, such as gene circuits, facilitating precise expression of acid resistance (AR) modules in a just-in-time and just-enough manner. RESULTS: In this study, we aimed to enhance the robustness and productivity of Escherichia coli, a workhorse for amino acid and organic acid production, in industrial fermentation under mild acidic conditions. We employed an Esa-type quorum sensing circuit to dynamically regulate the expression of an AR module (DsrA-Hfq) in a just-in-time and just-enough manner. Through careful engineering of the critical promoter PesaS and stepwise evaluation, we developed an optimal Esa-PBD(L) circuit that conferred upon an industrial E. coli strain SCEcL3 comparable lysine productivity and enhanced yield at pH 5.5 compared to the parent strain at pH 6.8. CONCLUSIONS: This study exemplifies the practical application of gene circuits in industrial environments, which present challenges far beyond those of well-controlled laboratory conditions.

Impact of AbaI mutation on virulence, biofilm development, and antibiotic susceptibility in Acinetobacter baumannii.

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.

A comparative genomic and phenotypic study of Vibrio cholerae model strains using hybrid sequencing.

Next-generation sequencing methods have become essential for studying bacterial biology and pathogenesis, often depending on high-quality, closed genomes. In this study, we utilized a hybrid sequencing approach to assemble the genome of C6706, a widely used Vibrio cholerae model strain. We present a manually curated annotation of the genome, enhancing user accessibility by linking each coding sequence to its counterpart in N16961, the first sequenced V. cholerae isolate and a commonly used reference genome. Comparative genomic analysis between V. cholerae C6706 and N16961 uncovered multiple genetic differences in genes associated with key biological functions. To determine whether these genetic variations result in phenotypic differences, we compared several phenotypes relevant to V. cholerae pathogenicity like genetic stability, acid sensitivity, biofilm formation and motility. Notably, V. cholerae N16961 exhibited greater motility and reduced biofilm formation compared to V. cholerae C6706. These phenotypic differences appear to be mediated by variations in quorum sensing and cyclic di-GMP signalling pathways between the strains. This study provides valuable insights into the regulation of biofilm formation and motility in V. cholerae.

The activity of the quorum sensing regulator HapR is modulated by the bacterial extracellular vesicle (BEV)-associated protein ObfA of Vibrio cholerae.

Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named outer membrane-associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.

New insights into pathogenic performances during peroxydisulfate composting: sources, pathways, and influencing factors.

Livestock manure treatment technology and composting and its products have been widely used in agricultural soil. However, little was known about the variations in the fate of pathogens and the factors affecting their pathogenic ability during this process, which posed threats to ecological safety and public health globally. This study used a metagenomic approach to profile the behaviors of pathogens during peroxydisulfate composting. Results showed that Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Burkholderia pseudomallei, and Mycobacterium tuberculosis were the main secretors of virulence factors (VFs) in composting system; their abundance and the virulence factor-related genes they carried were better downregulated under the role of peroxydisulfate. In addition, peroxydisulfate composting ensured the lower moisture, weakening the swimming mobility behavior of pathogens through suppressing the abundance of genes associated with flagellar formation, and impaired the communication between pathogens by regulating quorum sensing (QS)- and quorum quenching (QQ)-related genes. Moreover, reduced abundance of resistomes restricted pathogens disseminating infection. In summary, this study provided useful strategies in managing pathogen pathogenic ability during composting based on pathogenic source (pathogens), pathway (VFs), influencing factors (QS/QQ, physicochemical habitats), and resistomes.

Use of Rgg quorum-sensing machinery to create an innovative recombinant protein expression system in Streptococcus thermophilus.

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.