Search Results (42)

Quorum sensing (QS) represents a promising target for anti-virulence therapy; however, effective pharmacological intervention requires a detailed understanding of regulatory network architecture and environmental context. In Klebsiella pneumoniae, the orphan LuxR-type receptor SdiA lacks a cognate LuxI synthase and instead detects exogenous acyl-homoserine lactones (AHLs), positioning it as an inter-species signal integrator. Here, we demonstrate that SdiA functions as a context-dependent regulator whose impact on biofilm formation and virulence gene expression is gated by environmental AHL availability. Using isogenic ΔluxS, ΔsdiA, and ΔluxSΔsdiA mutants in a clinical bloodstream isolate, we show that under AHL-limited conditions, SdiA promotes baseline biofilm development, whereas in the presence of exogenous C6-HSL, it restrains excessive biofilm maturation. Two-way ANOVA confirmed significant genotype, treatment, and interaction effects, establishing that SdiA-mediated regulation is signal contingent. We further investigated the halogenated thiolactone meta-bromo-thiolactone (mBTL), previously described as a QS inhibitor in Pseudomonas aeruginosa. In K. pneumoniae, mBTL acts as a context-selective modulator rather than a simple inhibitor. Under AHL-limited conditions, mBTL phenocopied ΔsdiA, reducing biofilm formation and inducing overlapping transcriptional profiles. In contrast, under AHL-replete conditions, mBTL opposed SdiA-dependent gene expression, consistent with competitive antagonism of ligand-bound receptor. RNA-seq analysis revealed substantial concordance between ΔsdiA and WT + mBTL under AHL-free conditions, with the inversion of transcriptional directionality in the presence of C6-HSL. The findings redefine SdiA as a conditional quorum-sensing integrator and identify mBTL as a ligand-context-dependent modulator of LuxR-type signaling. Our results highlight the necessity of evaluating anti-virulence compounds across relevant signal environments and introduce receptor state-selective modulation as a strategic framework for targeting hybrid quorum-sensing systems in polymicrobial pathogens. Full article

Bacillus anthracis displays susceptibility to penicillin despite harboring a β-lactamase gene, a phenotype governed by the anti-sigma factor RsiP. While RsiP represses σ^P-dependent β-lactamase expression, its broader roles in physiology and virulence remain unclear. This study aimed to define the global regulatory functions of RsiP beyond antibiotic resistance. Deletion of rsiP significantly upregulated the nprR gene, which is an important quorum-sensing (QS) system regulator and enhanced protease secretion. The ΔrsiP mutant caused higher mortality in cellular and Galleria mellonella models and triggered elevated inflammatory cytokines (IL-6, IL-1β, TNF-α, MIP-2) in macrophages models. Surprisingly, in DBA/2 mice models, ΔrsiP was attenuated, with increased host survival and reduced bacterial loads. Competitive indices (CI) confirmed fitness defects in mice (spleen CI = 0.39; liver CI = 0.42). These defects were not due to altered oxidative stress tolerance but were attributed to impaired macrophage internalization of ΔrsiP spores, reducing early colonization. Our findings indicate that RsiP not only modulates β-lactam resistance but also influences extracellular protease activity and host adaptation. Full article

Lactulose, a valuable functional disaccharide with pharmaceutical and food applications, is efficiently synthesized via enzymatic isomerization of lactose. This study developed an integrated strategy combining protein engineering of cellobiose 2-epimerase (CsCE) from Caldicellulosiruptor saccharolyticus and process optimization to enhance lactulose production. A dual-track engineering approach—incorporating flexible loop modulation (residues 161–193) and structure-guided sequence alignment with N-acetyl-D-glucosamine-2-epimerase—enabled the creation of two superior mutants, R17Q/L184S and R17Q/S142T. The R17Q/L184S variant exhibited a 37% increase in crude enzyme activity, improved thermostability (half-life of 200 min at 80 °C), and enhanced substrate affinity (K_m reduced by 23.2%). R17Q/S142T achieved a 21% higher specific activity (24.08 U/mg), the highest among all variants. Structural and molecular dynamics analyses revealed that L184S enriched hydrogen bonding and hydrophobic interactions, improving structural rigidity, while S142T introduced allosteric regulation that facilitated catalytic efficiency. Under optimized conditions (70 °C, pH 7.5, 40% lactose, 20 U/mL enzyme, 3 h), lactulose yield reached 75.6% with >95% purity. This work demonstrates the successful application of synergistic enzyme engineering and process intensification for high-efficiency lactulose biosynthesis, providing viable candidates and system solutions for industrial-scale production. Full article

Visceral white spot disease caused by Pseudomonas plecoglossicida severely threatens marine aquaculture, highlighting the need for effective control strategies. To clarify the role of a novel small RNA, sRNA0024, in bacterial pathogenicity, we constructed an sRNA0024 deletion mutant (ΔsRNA0024) and compared its phenotype and virulence with those of the wild-type strain NZBD9. In vitro assays showed that deletion of sRNA0024 did not affect bacterial growth but significantly reduced biofilm formation and adhesion. In vivo infection experiments in orange-spotted grouper (Epinephelus coioides) demonstrated that the ΔsRNA0024 mutant had a 3.8-fold higher 50% lethal dose (LD₅₀), improved host survival, and milder splenic lesions than the wild type. Histopathology and host transcriptome analyses revealed weakened activation of complement–coagulation cascades, neutrophil extracellular traps, leukocyte migration, and inflammatory signaling pathways, indicating a lower-intensity immune response. Bacterial transcriptomics showed that deletion of sRNA0024 was associated with reduced luxR expression and attenuated quorum-sensing–associated virulence traits, supporting a possible role for this small RNA in modulating luxR expression and QS-related host immunopathology. These findings identify sRNA0024 as an important contributor to the virulence of P. plecoglossicida and highlight the sRNA0024–luxR module as a potential antivirulence target for controlling visceral white spot disease in aquaculture. Full article

Antibiotic misuse accelerates resistance dissemination via plasmid conjugation, but quorum sensing (QS) regulatory mechanisms remain undefined. Using Escherichia coli (E. coli) MG1655 conjugation models (RP4-7/EC600 plasmids), we demonstrate that long-chain acyl-homoserine lactones (C10/C12-HSL) enhance transfer frequency by up to 7.7-fold (200 μM C12-HSL; p < 0.001), while quorum-quenching by sub-inhibitory vanillin suppressed this effect by 95% (p < 0.0001). C12-HSL compromised membrane integrity via ompF upregulation (4-fold; p < 0.01) and conjugative pore assembly (trbBp upregulated by 1.38-fold; p < 0.05), coinciding with ROS accumulation (1.5-fold; p < 0.0001) and SOS response activation (recA upregulated by 1.68-fold; p < 0.001). Crucially, rpoS and rmf deletion mutants reduced conjugation by 65.5% and 55.8%, respectively (p < 0.001), exhibiting attenuated membrane permeability (≤65.5% reduced NPN influx; p < 0.0001), suppressed ROS (≤54% downregulated; p < 0.0001), and abolished transcriptional induction of conjugation/stress genes. Reciprocal RpoS–RMF (ribosomal hibernation factor) crosstalk was essential for AHL responsiveness, with deletions mutually suppressing expression (≤65.9% downregulated; p < 0.05). We establish a hierarchical mechanism wherein long-chain AHLs drive resistance dissemination through integrated membrane restructuring, stress adaptation, and RpoS–RMF-mediated genetic plasticity, positioning QS signaling as a viable target for curbing resistance spread. Full article

The mechanism by which quorum sensing (QS) enhances stress resistance in enterohemorrhagic Escherichia coli (E. coli) O157:H7 remains unclear. We employed optimized exogenous QS signal N-acyl-homoserinelactones (AHL) (100 μM 3-oxo-C6-AHL, 2 h) in EHEC O157:H7 strain EDL933, which was validated with endogenous yenI-derived AHL, to investigate QS-mediated protection against acid stress. RNA-seq transcriptomics identified key upregulated genes (e.g., rmf). Functional validation using isogenic rmf knockout mutants generated via λ-Red demonstrated abolished stress resistance and pan-stress vulnerability. Mechanistic studies employing qRT-PCR and stress survival assays established Ribosomal Hibernation Factor (RMF) as a non-redundant executor in a SdiA–RMF–RpoS axis, which activates ribosomal dormancy and SOS response to enhance EHEC survival under diverse stresses. For the first time, we define ribosomal hibernation as the core adaptive strategy linking QS to pathogen resilience, providing crucial mechanistic insights for developing EHEC control measures against foodborne threats. Full article

Infections often occur in complex niches consisting of multiple bacteria. Despite the increasing awareness, there is a fundamental gap in understanding which interactions govern microbial community composition. Pseudomonas aeruginosa is frequently isolated from monomicrobial and polymicrobial human infections. This pathogen forms polymicrobial infections with other ESKAPEE pathogens and defies eradication by conventional therapies. By analyzing the competition within co-cultures of P. aeruginosa and representative secondary pathogens that commonly co-infect patients, we demonstrate the antagonism of P. aeruginosa against other ESKAPEE pathogens and the contribution of this pathogen’s multiple quorum-sensing (QS) systems in these interactions. QS is a highly conserved bacterial cell-to-cell communication mechanism that coordinates collective gene expressions at the population level, and it is also involved in P. aeruginosa virulence. Using a collection of P. aeruginosa QS mutants of the three major systems, LasR/LasI, MvfR/PqsABCDE, and RhlR/RhlI, and mutants of several QS-regulated functions, we reveal that MvfR and, to a lesser extent, LasR and RhlR, control competition between P. aeruginosa and other microbes, possibly through their positive impact on pyoverdine, pyochelin, and phenazine genes. We show that MvfR inhibition alters competitive interspecies interactions and preserves the coexistence of P. aeruginosa with the ESKAPEE pathogens tested while disarming the pathogens’ ability to form biofilm and adhere to lung epithelial cells. Our results highlight the role of MvfR inhibition in modulating microbial competitive interactions across multiple species, while simultaneously attenuating virulence traits. These findings reveal the complexity and importance of QS in interspecies interactions and underscore the impact of the anti-virulence approach in microbial ecology and its importance for treating polymicrobial infections. Full article

Quorum sensing (QS) is a mechanism by which bacteria detect and respond to cell density, regulating collective behaviors. Burkholderia plantarii, the causal agent of rice seedling blight, employs the LuxIR-type QS system, common among Gram-negative bacteria, where LuxI-type synthase produces QS signals recognized by LuxR-type regulators to control gene expression. This study aimed to elucidate the QS mechanism in B. plantarii KACC18965. Through whole-genome analysis and autoinducer assays, the plaI gene, responsible for QS signal production, was identified. Motility assays confirmed that C8-homoserine lactone (C8-HSL) serves as the QS signal. Physiological experiments revealed that the QS-defective mutant exhibited reduced virulence, impaired swarming motility, and delayed biofilm formation compared to the wild type. Additionally, the QS mutant demonstrated weakened antibacterial activity against Escherichia coli and decreased phosphate solubilization. These findings indicate that QS in B. plantarii significantly influences various pathogenicity and survival traits, including motility, biofilm formation, antibacterial activity, and nutrient acquisition, highlighting the critical role of QS in pathogen virulence and adaptability. Full article

Pseudomonas aeruginosa is an opportunistic pathogen that usually causes chronic infections and even death in patients. The treatment of P. aeruginosa infection has become more challenging due to the prevalence of antibiotic resistance and the slow pace of new antibiotic development. Therefore, it is essential to explore non-antibiotic methods. A new strategy involves screening for drugs that target the quorum-sensing (QS) system. The QS system regulates the infection and drug resistance in P. aeruginosa. In this study, veratryl alcohol (VA) was found as an effective QS inhibitor (QSI). It effectively suppressed the expression of QS-related genes and the subsequent production of virulence factors under the control of QS including elastase, protease, pyocyanin and rhamnolipid at sub-inhibitory concentrations. In addition, motility activity and biofilm formation, which were correlated with the infection of P. aeruginosa, were also suppressed by VA. In vivo experiments demonstrated that VA could weaken the pathogenicity of P. aeruginosa in Chinese cabbage, Drosophila melanogaster, and Caenorhabditis elegans infection models. Molecular docking, combined with QS quintuple mutant infection analysis, identified that the mechanism of VA could target the LasR protein of the las system mainly. Moreover, VA increased the susceptibility of P. aeruginosa to conventional antibiotics of tobramycin, kanamycin and gentamicin. The results firstly demonstrate that VA is a promising QSI to treat infections caused by P. aeruginosa. Full article

Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicates its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 34-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. The increased specificity of E380Q/S387L not only enhances its ability to break down pro-immunogenic peptides but also positions this enzyme variant as a promising candidate for oral therapy for celiac disease. Full article

Methanethiol (MeSH) and dimethyl sulfide (DMS) are important volatile organic sulfur compounds involved in atmospheric chemistry and climate regulation. However, little is known about the metabolism of these compounds in the ubiquitous marine vibrios. Here, we investigated MeSH/DMS production and whether these processes were regulated by quorum-sensing (QS) systems in Vibrio harveyi BB120. V. harveyi BB120 exhibited strong MeSH production from methionine (Met) (465 nmol mg total protein⁻¹) and weak DMS production from dimethylsulfoniopropionate (DMSP) cleavage. The homologs of MegL responsible for MeSH production from L-Met widely existed in vibrio genomes. Using BB120 and its nine QS mutants, we found that the MeSH production was regulated by HAI-1, AI-2 and CAI-1 QS pathways, as well as the luxO gene located in the center of this QS cascade. The regulation role of HAI-1 and AI-2 QS systems in MeSH production was further confirmed by applying quorum-quenching enzyme MomL and exogenous autoinducer AI-2. By contrast, the DMS production from DMSP cleavage showed no significant difference between BB120 and its QS mutants. Such QS-regulated MeSH production may help to remove excess Met that can be harmful for vibrio growth. These results emphasize the importance of QS systems and the MeSH production process in vibrios. Full article

Pseudomonas aeruginosa (PAO1) is an opportunistic pathogen, lethal in immunocompromised individuals. The clinical management of PAO1 infections still depends deeply on antibiotic therapy. However, this therapy has been alarmingly overpowered by growing bacterial resistance mechanisms over the years. One of these bacterial mechanisms is quorum sensing (QS). QS is involved in the production of biofilm, rhamnolipids and pyocyanin, among other factors. The present study aimed to study the effect of the mutations in the genes of rubredoxin (Rub A1 and Rub A2) and glutaredoxin (GLRx) in the production of virulence traits and susceptibility of PAO1 to the antibiotic ciprofloxacin (CIP) and to infection by a phage cocktail. Rub A1, Rub A2, and GLRx showed a decrease in the expression of genes lasI, lasR, mvfR, and rpsL when compared to the wild type, PAO1. Rub A1 and Rub A2 also showed a decrease in the expression of the gene pqsA, while the mutant GLRx showed an increase of over 200% in expression compared to PAO1. The biofilm produced by the mutants Rub A1, Rub A2, and GLRx increased more than 1.5 times in comparison to PAO1, with statistical significance (p < 0.0001). In the viability assay, the mutant strain Rub A2 was the most susceptible to ciprofloxacin in both concentrations tested (p < 0.0001). The production of proteases increased in the mutant strains when compared to PAO1 (p < 0.05). However, there was a decrease in the production of rhamnolipids and pyocyanins in the mutant strains. In the phage assay, we could perceive a reduction in the growth of the mutant strains when compared to PAO1. Additionally, after the addition of the phages, all the strains showed susceptibility to the phage assay (p < 0.0001), observed in the decrease in the absorbance values. These results may highlight the relevance of the genes Rub A1, Rub A2, and GLRX in the proliferation and treatment of infections with PAO1. Overall, this study gives preliminary insights into how gene expression may be helpful in strategies to overcome antibiotic resistance. Full article

This study aimed to investigate the effect of reduced expression of the luxR gene on the virulence of Pseudomonas plecoglossicida and the immune response of Epinephelus coioides. To achieve this, RNA interference (RNAi) was used to silence the luxR gene, and the pathogenicity of wild-type and luxR-RNAi strains of P. plecoglossicida, as well as the immune response of Epinephelus coioides to the infection of these two strains, were compared. The mutant strain with the highest silencing efficiency of 70.1% was selected for subsequent analysis. Silencing the luxR gene in the mutant strain resulted in a significant 30% reduction in mortality rates in artificially infected Epinephelus coioides compared to the wild-type strain. Transcriptome analysis revealed that the host transcriptome, particularly in the spleens of infected Epinephelus coioides, was markedly altered by the silencing of the luxR gene in the mutant strain. Tilapia infected with the luxR-RNAi strain exhibited altered immune defenses, with changes in gene expression primarily in the NOD-like receptor (NLR) signaling pathway. These results suggest that the luxR gene plays a crucial role in the host’s resistance to pathogen invasion, and reducing its expression could decrease quorum sensing (QS) signals while increasing the expression of the IL-1β gene in the host’s NLR pathway. This effect may lead to a pro-inflammatory response that enhances the immune response to infection. Further investigation of these mechanisms may lead to innovative approaches to treating bacterial infections. Full article

Shikimate is a valuable chiral precursor for synthesizing oseltamivir (Tamiflu^®) and other chemicals. High production of shikimate via microbial fermentation has attracted increasing attention to overcome the unstable and expensive supply of shikimate extracted from plant resources. The current cost of microbial production of shikimate via engineered strains is still unsatisfactory, and thus more metabolic strategies need to be investigated to further increase the production efficiency. In this study, we first constructed a shikimate E. coli producer through the application of the non-phosphoenolpyruvate: carbohydrate phosphotransferase system (non-PTS) glucose uptake pathway, the attenuation of the shikimate degradation metabolism, and the introduction of a mutant of feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase. Inspired by the natural presence of bifunctional 3-dehydroquinate dehydratase (DHD)-shikimate dehydrogenase (SDH) enzyme in plants, we then designed an artificial fusion protein of DHD-SDH to decrease the accumulation of the byproduct 3-dehydroshikimate (DHS). Subsequently, a repressed shikimate kinase (SK) mutant was selected to promote shikimate accumulation without the supplementation of expensive aromatic substances. Furthermore, EsaR-based quorum sensing (QS) circuits were employed to regulate the metabolic flux distribution between cell growth and product synthesis. The final engineered strain dSA10 produced 60.31 g/L shikimate with a yield of 0.30 g/g glucose in a 5 L bioreactor. Full article

DSPAα1 is a potent rude thrombolytic protein with high medicative value. DSPAα1 has two natural N-glycan sites (N153Q-S154-S155, N398Q-K399-T400) that may lead to immune responses when administered in vivo. We aimed to study the effect of its N-glycosylation sites on DSPAα1 in vitro and in vivo by mutating these N-glycosylation sites. In this experiment, four single mutants and one double mutant were predicted and expressed in Pichia pastoris. When the N398Q-K399-T400 site was mutated, the fibrinolytic activity of the mutant was reduced by 75%. When the N153Q-S154-S155 sites were inactivated as described above, the plasminogen activating activity of its mutant was reduced by 40%, and fibrin selectivity was significantly reduced by 21-fold. The introduction of N-glycosylation on N184-G185-A186T and K368N-S369-S370 also considerably reduced the activity and fibrin selectivity of DSPAα1. The pH tolerance and thermotolerance of all mutants did not change significantly. In vivo experiments also confirmed that N-glycosylation mutations can reduce the safety of DSPAα1, lead to prolonged bleeding time, non-physiological reduction of coagulation factor (α2-AP, PAI) concentration, and increase the risk of irregular bleeding. This study ultimately demonstrated the effect of N-glycosylation mutations on the activity and safety of DSPAα1. Full article