RESUMEN
Aeromonas salmonicida is an opportunistic pathogen with relevance for aquaculture. Fish epithelia are covered by a mucus layer, composed mainly by highly glycosylated mucins, which are the first point of contact between fish and pathogens. Quorum sensing (QS), a bacterial communication mechanism through secreted autoinducer signals that governs gene expression, influences bacterial growth and virulence. The main A. salmonicida autoinducers are mediated by the luxS and asaI genes, corresponding to inter- and intraspecies communication, respectively. The aim of this study was to determine the effect of the mucins that pathogens encounter during colonization of the gill and skin on A. salmonicida QS. We found that expression of A. salmonicida asaI, but not luxS, was increased after culture at 20 °C compared to 10 °C. Rainbow trout gill and skin mucins up-regulated asaI expression 2-fold but down-regulated luxS 10-fold. The downregulation of luxS was reflected by a reduction in autoinducer-2 secretion. Mucins isolated from skin had a stronger inhibitory effect than mucins isolated from gills on both luxS expression and A1-2 secretion, consistent with a higher relative abundance of N-Acetylneuraminic acid on skin mucins than on gill mucins. Reduction of AI-2 production by mucins or luxS-deletion lead to a reduced A. salmonicida auto-aggregation. Furthermore, after colonization of the gill, luxS was down regulated whereas asaI expression was upregulated. Both in vivo and in vitro, the expression of luxS and asaI were thus differentially regulated, frequently in an inverse manner. The strong AI-2 inhibiting effect of the skin mucins is likely part of the mucin-based defense against pathogens.
Asunto(s)
Aeromonas salmonicida , Homoserina , Mucinas , Oncorhynchus mykiss , Percepción de Quorum , Animales , Oncorhynchus mykiss/inmunología , Aeromonas salmonicida/fisiología , Mucinas/genética , Mucinas/metabolismo , Homoserina/análogos & derivados , Liasas de Carbono-Azufre/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Proteínas Bacterianas/genética , Lactonas , Piel/inmunología , Piel/microbiología , Branquias/inmunología , Branquias/metabolismoRESUMEN
l-Homoserine is a promising C4 platform compound used in the agricultural, cosmetic, and pharmaceutical industries. Numerous works have been conducted to engineer Escherichia coli to be an excellent l-homoserine producer, but it is still unable to meet the industrial-scale demand. Herein, we successfully engineered a plasmid-free and noninducible E. coli strain with highly efficient l-homoserine production through balancing AspC and AspA synthesis pathways. First, an initial strain was constructed by increasing the accumulation of the precursor oxaloacetate and attenuating the organic acid synthesis pathway. To remodel the carbon flux toward l-aspartate, a balanced route prone to high yield based on TCA intensity regulation was designed. Subsequently, the main synthetic pathway and the cofactor system were strengthened to reinforce the l-homoserine synthesis. Ultimately, under two-stage DO control, strain HSY43 showed 125.07 g/L l-homoserine production in a 5 L fermenter in 60 h, with a yield of 0.62 g/g glucose and a productivity of 2.08 g/L/h. The titer, yield, and productivity surpassed the highest reported levels for plasmid-free strains in the literature. The strategies adopted in this study can be applied to the production of other l-aspartate family amino acids.
Asunto(s)
Escherichia coli , Homoserina , Ingeniería Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Homoserina/metabolismo , Homoserina/análogos & derivados , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Aspártico/metabolismo , Fermentación , Plásmidos/genética , Plásmidos/metabolismoRESUMEN
Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages. IMPORTANCE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.
Asunto(s)
Percepción de Quorum , Vibrio alginolyticus , Vibrio alginolyticus/virología , Vibrio alginolyticus/fisiología , Bacteriófagos/fisiología , Bacteriófagos/genética , Homoserina/análogos & derivados , Homoserina/metabolismo , Cápsulas Bacterianas/metabolismo , Podoviridae/genética , Podoviridae/fisiología , Biopelículas/crecimiento & desarrollo , Polisacáridos Bacterianos/metabolismoRESUMEN
Pseudomonas aeruginosa (P. aeruginosa) biofilm formation is a crucial cause of enhanced antibiotic resistance. Quorum sensing (QS) is involved in regulating biofilm formation; QS inhibitors block the QS signaling pathway as a new strategy to address bacterial resistance. This study investigated the potential and mechanism of L-HSL (N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide) as a QS inhibitor for P. aeruginosa. The results showed that L-HSL effectively inhibited the biofilm formation and dispersed the pre-formed biofilm of P. aeruginosa. The production of extracellular polysaccharides and the motility ability of P. aeruginosa were suppressed by L-HSL. C. elegans infection experiment showed that L-HSL was non-toxic and provided protection to C. elegans against P. aeruginosa infection. Transcriptomic analysis revealed that L-HSL downregulated genes related to QS pathways and biofilm formation. L-HSL exhibits a promising potential as a therapeutic drug for P. aeruginosa infection. KEY POINTS: ⢠Chemical synthesis of N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide, named L-HSL. ⢠L-HSL does not generate survival pressure on the growth of P. aeruginosa and can inhibit the QS system. ⢠KEGG enrichment analysis found that after L-HSL treatment, QS-related genes were downregulated.
Asunto(s)
4-Butirolactona , Biopelículas , Caenorhabditis elegans , Pseudomonas aeruginosa , Percepción de Quorum , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/genética , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Percepción de Quorum/efectos de los fármacos , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/microbiología , Animales , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , 4-Butirolactona/metabolismo , Antibacterianos/farmacología , Perfilación de la Expresión Génica , Homoserina/análogos & derivados , Homoserina/metabolismo , Homoserina/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacosRESUMEN
In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.
Asunto(s)
Proteínas Bacterianas , Biopelículas , Liasas de Carbono-Azufre , Regulación Bacteriana de la Expresión Génica , Homoserina , Percepción de Quorum , Biopelículas/crecimiento & desarrollo , Liasas de Carbono-Azufre/genética , Virulencia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Homoserina/análogos & derivados , Mutación , Factores de Virulencia/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Animales , Salmonella/patogenicidad , Salmonella/genéticaRESUMEN
BACKGROUND: Imbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation. OBJECTIVE: This study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells. METHODS: Pathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1ß (IL-1ß), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence. RESULTS: AI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1ß and IL-6 mRNA expression (P < 0.05). CONCLUSION: AI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.
Asunto(s)
Enterocolitis Necrotizante , Metaloproteinasa 3 de la Matriz , Receptor PAR-2 , Transducción de Señal , Animales , Humanos , Ratones , Animales Recién Nacidos , Línea Celular , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/patología , Enterocolitis Necrotizante/tratamiento farmacológico , Enterocolitis Necrotizante/metabolismo , Homoserina/análogos & derivados , Homoserina/farmacología , Mucosa Intestinal/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/inmunología , Intestinos/patología , Intestinos/efectos de los fármacos , Lactonas/farmacología , Lipopolisacáridos , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Ratones Endogámicos C57BL , Ocludina/metabolismo , Ocludina/genética , Receptor PAR-2/metabolismo , Receptor PAR-2/genética , Transducción de Señal/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genéticaRESUMEN
Bacterial biofilms pose significant a public health risk as an environmental reservoir for opportunistic aquatic bacterial pathogens. Understanding the interspecies roles of complex bacterial biofilms under different stimuli and regulatory mechanisms of stress responses is the key to controlling their dissemination. Herein, two-species mixture (TSM) biofilms (Staphylococcus aureus and Pseudomonas aeruginosa) were constructed in a flowthrough reactor. Compared with the single-species biofilms, the TSM biofilm had higher growth activity to reach maturity faster, forming a staggered community structure. Moreover, the TSM biofilm exhibited greatly improved resistance to different antibiotics (16-128 times higher), especially to those that act on protein synthesis and cell membrane integrity, when compared to single planktonic microorganisms. In the presence of stimuli, photocatalysis effectively inactivated the TSM biofilm within 10 h, a 4-fold shorter inactivation time compared to UVC irradiation. In addition, photocatalysis effectively depleted the extracellular polymers of the TSM biofilm and inhibited secretion of their interspecies quorum sensing signaling molecule autoinducer-2 (AI-2). However, the expression of AI-2 induced related virulence factors, and biofilm growth-related genes were initially up-regulated 3 - 10 fold for the TSM biofilm within the first 2 - 4 h of photocatalysis, followed by significant down-regulation. Furthermore, the addition of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione effectively delayed the photocatalytic inactivation efficiency of the TSM biofilm compared to the control. These results suggest that photocatalysis can effectively inactivate biofilms by inhibiting interspecies cooperation by quenching AI-2 in the TSM biofilm. This work sheds light on controlling biofilms in public health engineering systems.
Asunto(s)
Biopelículas , Pseudomonas aeruginosa , Percepción de Quorum , Staphylococcus aureus , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/fisiología , Catálisis , Antibacterianos/farmacología , Lactonas/metabolismo , Homoserina/análogos & derivados , Microbiología del Agua , Rayos UltravioletaRESUMEN
Bacteria utilize intercellular communication to orchestrate essential cellular processes, adapt to environmental changes, develop antibiotic tolerance, and enhance virulence. This communication, known as quorum sensing (QS), is mediated by the exchange of small signalling molecules called autoinducers. AI-2 QS, regulated by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase), acts as a universal intercellular communication mechanism across gram-positive and gram-negative bacteria and is crucial for diverse bacterial processes. In this study, we demonstrated that in Streptococcus suis (S. suis), a notable zoonotic pathogen, AI-2 QS enhances galactose utilization, upregulates the Leloir pathway for capsular polysaccharide (CPS) precursor production, and boosts CPS synthesis, leading to increased resistance to macrophage phagocytosis. Additionally, our molecular docking and dynamics simulations suggest that, similar to S. pneumoniae, FruA, a fructose-specific phosphoenolpyruvate phosphotransferase system prevalent in gram-positive pathogens, may also function as an AI-2 membrane surface receptor in S. suis. In conclusion, our study demonstrated the significance of AI-2 in the synthesis of galactose metabolism-dependent CPS in S. suis. Additionally, we conducted a preliminary analysis of the potential role of FruA as a membrane surface receptor for S. suis AI-2.
Asunto(s)
Galactosa , Percepción de Quorum , Streptococcus suis , Streptococcus suis/fisiología , Galactosa/metabolismo , Percepción de Quorum/fisiología , Virulencia , Animales , Cápsulas Bacterianas/metabolismo , Lactonas/metabolismo , Infecciones Estreptocócicas/veterinaria , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/inmunología , Homoserina/análogos & derivados , Homoserina/metabolismo , Polisacáridos Bacterianos/metabolismoRESUMEN
The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.
Asunto(s)
4-Butirolactona , Angiopoyetina 2 , Homoserina , Células Endoteliales de la Vena Umbilical Humana , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Angiopoyetina 2/metabolismo , Angiopoyetina 2/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Homoserina/análogos & derivados , Homoserina/farmacología , Homoserina/metabolismo , Cadherinas/metabolismo , Cadherinas/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Transducción de Señal/efectos de los fármacos , Antígenos CD/metabolismo , Antígenos CD/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 1/genéticaRESUMEN
Fusobacterium nucleatum (F. nucleatum) is closely correlated with tumorigenesis in colorectal cancer (CRC). We aimed to investigate the effects of host norepinephrine on the carcinogenicity of F. nucleatum in CRC and reveal the underlying mechanism. The results revealed that both norepinephrine and bacterial quorum sensing (QS) molecule auto-inducer-2 (AI-2) were positively associated with the progression of F. nucleatum related CRC (p < 0.01). In vitro studies, norepinephrine induced upregulation of QS-associated genes and promoted the virulence and proliferation of F. nucleatum. Moreover, chronic stress significantly increased the colon tumour burden of ApcMin/+ mice infected with F. nucleatum (p < 0.01), which was decreased by a catecholamine inhibitor (p < 0.001). Our findings suggest that stress-induced norepinephrine may promote the progression of F. nucleatum related CRC via bacterial QS signalling. These preliminary data provide a novel strategy for the management of pathogenic bacteria by targeting host hormones-bacterial QS inter-kingdom signalling.
Asunto(s)
Neoplasias Colorrectales , Fusobacterium nucleatum , Norepinefrina , Percepción de Quorum , Transducción de Señal , Percepción de Quorum/efectos de los fármacos , Fusobacterium nucleatum/patogenicidad , Fusobacterium nucleatum/efectos de los fármacos , Fusobacterium nucleatum/fisiología , Animales , Neoplasias Colorrectales/microbiología , Norepinefrina/farmacología , Ratones , Humanos , Progresión de la Enfermedad , Infecciones por Fusobacterium/microbiología , Virulencia , Homoserina/análogos & derivados , Homoserina/metabolismo , Ratones Endogámicos C57BL , Masculino , LactonasRESUMEN
Symbiotic microorganisms play critical ecophysiological roles that facilitate the maintenance of coral health. Currently, information on the gene and protein pathways contributing to bleaching responses is lacking, including the role of autoinducers. Although the autoinducer AI-1 is well understood, information on AI-2 is insufficient. Here, we observed a 3.7-4.0 times higher abundance of the AI-2 synthesis gene luxS in bleached individuals relative to their healthy counterparts among reef-building coral samples from the natural environment. Laboratory tests further revealed that AI-2 contributed significantly to an increase in coral bleaching, altered the ratio of potential probiotic and pathogenic bacteria, and suppressed the antiviral activity of specific pathogenic bacteria while enhancing their functional potential, such as energy metabolism, chemotaxis, biofilm formation and virulence release. Structural equation modeling indicated that AI-2 influences the microbial composition, network structure, and pathogenic features, which collectively contribute to the coral bleaching status. Collectively, our results offer novel potential strategies for coral conservation based on a signal manipulation approach.
Asunto(s)
Antozoos , Homeostasis , Percepción de Quorum , Simbiosis , Antozoos/microbiología , Antozoos/fisiología , Animales , Homoserina/análogos & derivados , Homoserina/metabolismo , Arrecifes de Coral , Lactonas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
Acetate, a promising yet underutilized carbon source for biological production, was explored for the efficient production of homoserine and threonine in Escherichia coli W. A modular metabolic engineering approach revealed the crucial roles of both acetate assimilation pathways (AckA/Pta and Acs), optimized TCA cycle flux and glyoxylate shunt activity, and enhanced CoA availability, mediated by increased pantothenate kinase activity, for efficient homoserine production. The engineered strain W-H22/pM2/pR1P exhibited a high acetate assimilation rate (5.47 mmol/g cell/h) and produced 44.1 g/L homoserine in 52 h with a 53% theoretical yield (0.18 mol/mol) in fed-batch fermentation. Similarly, strain W-H31/pM2/pR1P achieved 45.8 g/L threonine in 52 h with a 65% yield (0.22 mol/mol). These results represent the highest reported levels of amino acid production using acetate, highlighting its potential as a valuable and sustainable feedstock for biomanufacturing.
Asunto(s)
Acetatos , Escherichia coli , Homoserina , Ingeniería Metabólica , Treonina , Escherichia coli/genética , Escherichia coli/metabolismo , Treonina/biosíntesis , Treonina/metabolismo , Treonina/genética , Acetatos/metabolismo , Homoserina/metabolismo , Homoserina/análogos & derivados , Homoserina/genética , Homoserina/biosíntesis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Industrial wastewater is a major environmental concern due to its high copper content, which poses significant toxicity to microbial life. Autoinducer-2 (AI-2) can participate in the inter- and intra-species communication and regulate the physiological functions of different bacterial species by producing AI-2 signal molecules. However, there are few research reports on the luxS gene and lsr operon functions for AI-2 in bacteria with a certain tolerance to copper. This study delves into the potential of quorum sensing mechanisms, particularly the AI-2 system, for enhancing microbial resistance to copper toxicity in Klebsiella michiganensis (KM). We detail the critical roles of the luxS gene in AI-2 synthesis and the lsr operon in AI-2 uptake, demonstrating their collective impact on enhancing copper resistance. Our findings show that mutations in the lsr operon, alongside the knockout of the luxS gene in KM strain (KMΔluxSΔlsr), significantly impair the strain's motility (p < 0.0001) and biofilm formation (p < 0.01), underscoring the operon's role in AI-2 transport. These genetic insights are pivotal for developing bioremediation strategies aimed at mitigating copper pollution in wastewater. By elucidating the mechanisms through which KM modulates copper resistance, this study highlights the broader ecological significance of leveraging microbial quorum sensing pathways for sustainable wastewater management.
Asunto(s)
Proteínas Bacterianas , Liasas de Carbono-Azufre , Cobre , Klebsiella , Operón , Percepción de Quorum , Cobre/toxicidad , Percepción de Quorum/efectos de los fármacos , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Klebsiella/genética , Klebsiella/efectos de los fármacos , Klebsiella/metabolismo , Homoserina/análogos & derivados , Homoserina/metabolismo , Lactonas/metabolismoRESUMEN
Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogenic bacterium that causes huge economic losses to the pig farming industry and considerably threatens human health. The quorum sensing (QS) system plays a crucial role in the survival and pathogenesis of pathogenic bacteria. Hence, it is a viable approach to prevent ExPEC infection by compromising the QS system, particularly the LuxS/AI-2 system. In this study, we investigated the effects of baicalin on the LuxS/AI-2 system of ExPEC. Baicalin at concentrations of 25, 50, and 100 µg/mL significantly diminished the survival ability of ExPEC in hostile environments and could inhibit the biofilm formation and autoagglutination ability in ExPEC. Moreover, baicalin dose-dependently decreased the production of AI-2 and down-regulated the expression level of luxS in PCN033. These results suggest that baicalin can weaken the virulence of PCN033 by inhibiting the LuxS/AI-2 system. After the gene luxS was deleted, AI-2 production in PCN033 was almost completely eliminated, similar to the effect of baicalin on the production of AI-2 in PCN033. This indicates that baicalin reduced the production of AI-2 by inhibiting the expression level of luxS in ExPEC. In addition, the animal experiment further showed the potential of baicalin as a LuxS/AI-2 system inhibitor to prevent ExPEC infection. This study highlights the potential of baicalin as a natural quorum-sensing inhibitor for therapeutic applications in preventing ExPEC infection by targeting the LuxS/AI-2 system.
Asunto(s)
Proteínas Bacterianas , Liasas de Carbono-Azufre , Escherichia coli Patógena Extraintestinal , Flavonoides , Homoserina , Homoserina/análogos & derivados , Percepción de Quorum , Percepción de Quorum/efectos de los fármacos , Flavonoides/farmacología , Animales , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Porcinos , Virulencia/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Homoserina/metabolismo , Escherichia coli Patógena Extraintestinal/efectos de los fármacos , Escherichia coli Patógena Extraintestinal/patogenicidad , Escherichia coli Patógena Extraintestinal/genética , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Lactonas/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/tratamiento farmacológicoRESUMEN
Bacteria produce and release small signal molecules, autoinducers, as an indicator of their cell density. The system, called a quorum-sensing (QS) system, is used to control not only virulence factors but also antibiotic production, sporulation, competence, and biofilm formation in bacteria. Different from antibiotics, QS inhibitors are expected to specifically repress the virulence factors in pathogenic bacteria without inhibiting growth or bactericidal effects. Therefore, since QS inhibitors have little risk of antibiotic-resistant bacteria emergence, they have been proposed as promising anti-bacterial agents. In the present study, we aimed to find new QS inhibitors that prohibit the signaling cascade of autoinducer 3 (AI-3) recognized by a QseCB two-component system that regulates some virulence factors of pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica subsp. enterica serovar Typhimurium. We have established the method for QS-inhibitor screening using a newly constructed plasmid pLES-AQSA. E. coli DH5α transformed with the pLES-AQSA can produce ß-galactosidase that converts 5-bromo-4-chloro-3-indolyl ß-d-galactopyranoside (X-gal) into blue pigment (5-bromo-4-chloro-indoxyl) under the control of the QseCB system. By screening, Heyndrickxia coagulans (formerly Bacillus coagulans) 29-2E was found to produce an exopolysaccharide (EPS)-like water-soluble polymer that prohibits QseCB-mediated ß-galactosidase production without antibacterial activities. Further, the simultaneous injection of the 29-2E strain significantly improves the survival rate of Salmonella Typhimurium-infected silkworm larvae (from 0% to 83.3%), suggesting that the substance may be a promising inhibitor against the virulence of pathogens without risk of the emergence of antibiotic-resistant bacteria.
Asunto(s)
Percepción de Quorum , Salmonella typhimurium , Percepción de Quorum/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Virulencia , Bacillus/metabolismo , Antibacterianos/farmacología , Lactonas/farmacología , Lactonas/metabolismo , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Escherichia coli Enterohemorrágica/efectos de los fármacos , Escherichia coli Enterohemorrágica/patogenicidad , Escherichia coli Enterohemorrágica/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Homoserina/análogos & derivadosRESUMEN
Cutibacterium acnes is an opportunistic pathogen in acne vulgaris. C. acnes produces autoinducer-2 (AI-2ï¼, a signaling molecule used for communication known as quorum sensing (QSï¼. In C. acnes, QS reportedly upregulates biofilm formation leading to resistance against bactericidal agents. In this study, we analyzed how heparinoid affected QS and biofilm formation of the opportunistic pathogen C. acnes. We also verified whether heparinoid would suppress biofilm formation and enhance the efficacy of the bactericidal agent 4-isopropyl-3-methylphenol (IPMP) against C. acnes biofilms. We ran an AI-2 bioassay using Vibrio harveyi ATCC BBA-1121. Heparinoid exhibited inhibitory activity against AI-2 at concentrations of 0.003-0.005%, suggesting an AI-2 analog-derived or C. acnes culture supernatant-derived inhibition of the AI-2 activity. To evaluate how heparinoid suppresses biofilm formation in C. acnes, we completed a biofilm assay in 96-well plates. We also evaluated the bactericidal activity of IPMP against the C. acnes biofilm prepared with or without heparinoid. Heparinoid inhibited C. acnes biofilm formation and IPMP bactericidal efficacy increased upon heparinoid-mediated suppression of biofilm formation. In this study, we clarified that heparinoid inhibits the AI-2-mediated QS of C. acnes, thereby suppressing biofilm formation and increasing IPMP bactericidal efficacy, potentially suppressing acne vulgaris.
Asunto(s)
Acné Vulgar , Heparinoides , Homoserina/análogos & derivados , Lactonas , Humanos , Percepción de Quorum , Heparinoides/farmacología , Biopelículas , Antibacterianos/farmacología , Acné Vulgar/tratamiento farmacológicoRESUMEN
OBJECTIVE: The aim of this study was to investigate the association between autoinducer-2 (AI-2) of oral microbial flora and the alveolar bone destruction in periodontitis to determine if AI-2 may have the potential that monitor periodontitis and predict bone loss. BACKGROUND: Plaque biofilm was the initiating factor of periodontitis and the essential factor of periodontal tissue destruction. The formation of biofilms depended on the complex regulation of the quorum sensing (QS) system, in which bacteria could sense changes in surrounding bacterial density by secreting the autoinducer (AI) to regulate the corresponding physiological function. Most oral bacteria also communicated with each other to form biofilms administrating the QS system, which implied that the QS system of periodontal pathogens was related to periodontitis, but the specific relationship was unknown. METHOD: We collected the gingival crevicular fluid (GCF) samples and measured the concentration of AI-2 in samples using the Vibrio harveyi BB180 bioluminescent-reporter system. To explore the interaction between AI-2 and bone metabolism, we utilized AI-2 purified from Fusobacterium nucleatum to investigate the impact of F. nucleatum AI-2 on osteoclast differentiation. Moreover, we constructed murine periodontitis models and multi-species biofilm models to study the association between AI-2 and periodontal disease progression. RESULTS: The AI-2 concentration in GCF samples increased along with periodontal disease progression (p < .0001). F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner. In the periodontitis mice model, the CEJ-ABC distance in the F. nucleatum AI-2 treatment group was higher than that in the simple ligation group (p < .01), and the maxilla of the mice in the group exhibited significantly lower BMD and BV/TV values (p < .05). CONCLUSIONS: We demonstrated that the AI-2 concentration varied with the alveolar bone destruction in periodontitis, and it may have the potential for screening periodontitis. F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner and aggravated bone loss.
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Pérdida de Hueso Alveolar , Biopelículas , Fusobacterium nucleatum , Homoserina , Lactonas , Periodontitis , Pérdida de Hueso Alveolar/microbiología , Pérdida de Hueso Alveolar/metabolismo , Periodontitis/microbiología , Animales , Homoserina/análogos & derivados , Homoserina/metabolismo , Biopelículas/crecimiento & desarrollo , Ratones , Humanos , Líquido del Surco Gingival/microbiología , Líquido del Surco Gingival/química , Masculino , Modelos Animales de Enfermedad , Osteoclastos , Percepción de Quorum , Femenino , Adulto , Diferenciación Celular , Persona de Mediana Edad , Microtomografía por Rayos XRESUMEN
Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.
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Percepción de Quorum , Streptococcus equi , Humanos , Ratones , Animales , Streptococcus equi/genética , Streptococcus equi/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Homoserina/metabolismo , Lactonas/metabolismo , BiopelículasRESUMEN
Efficient quorum sensing (QS) response is the premise for recovering the activities of stored aerobic granular sludge (AGS). This study aims to explore the crosstalk between the secondary messenger and the N-acylated-homoserine lactones (AHLs) to yield protein-rich granules efficiently from stored AGS by enhancing its QS efficiency selectively. 80 nmol/L cyclic diguanylate (c-di-GMP) with 20 nmol/L AHLs could increase the activity of isocitrate lyase activity (ICD) by 89 % and isocitrate dehydrogenase activity (ICDHc) by 113.5 %, to accelerate the tricarboxylic acid (TCA) cycle for yielding excess proteins by 166.4 %. In contrast, 80 nmol/L autoinducer-2 (AI-2) with 20 nmol/L AHLs could increase the activities of ICD and ICDHc by 485 % and 54.5 %, respectively, accelerating the glyoxylate (GCA) cycle to activate fat acid synthesis for stimulating polysaccharides (PS) secretion by 137.9 %. The strategy with c-di-GMP successfully recovers the refrigerated-stored and dried-stored AGS into proteins-rich AGS, with enriched functional strains for the PN secretion.
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Acil-Butirolactonas , Homoserina/análogos & derivados , Aguas del Alcantarillado , Acil-Butirolactonas/metabolismo , Percepción de Quorum , Lactonas , Sistemas de Mensajero SecundarioRESUMEN
For several decades, the formation of microbial self-aggregates, known as granules, has been extensively documented in the context of anaerobic digestion. However, current understanding of the underlying microbial-associated mechanisms responsible for this phenomenon remains limited. This study examined morphological and biochemical changes associated with cell aggregation in model co-cultures of the syntrophic propionate oxidizing bacterium Syntrophobacterium fumaroxidans and hydrogenotrophic methanogens, Methanospirillum hungatei or Methanobacterium formicicum. Formerly, we observed that when syntrophs grow for long periods with methanogens, cultures tend to form aggregates visible to the eye. In this study, we maintained syntrophic co-cultures of S. fumaroxidans with either M. hungatei or M. formicicum for a year in a fed-batch growth mode to stimulate aggregation. Millimeter-scale aggregates were observed in both co-cultures within the first 5 months of cultivation. In addition, we detected quorum sensing molecules, specifically N-acyl homoserine lactones, in co-culture supernatants preceding the formation of macro-aggregates (with diameter of more than 20 µm). Comparative transcriptomics revealed higher expression of genes related to signal transduction, polysaccharide secretion and metal transporters in the late-aggregation state co-cultures, compared to the initial ones. This is the first study to report in detail both biochemical and physiological changes associated with the aggregate formation in syntrophic methanogenic co-cultures. KEYPOINTS: ⢠Syntrophic co-cultures formed mm-scale aggregates within 5 months of fed-batch cultivation. ⢠N-acyl homoserine lactones were detected during the formation of aggregates. ⢠Aggregated co-cultures exhibited upregulated expression of adhesins- and polysaccharide-associated genes.