RESUMO
Aim: To search for potential inhibitors to homoserine dehydrogenase (HSD) in Paracoccidioides brasiliensis the causative agent of paracoccidioidomycosis, an infection with a high mortality rate in Brazil.Materials & methods: The enzyme was modeled and used in the virtual screening of the compounds. The library was first screened by the Autodock, in which 66 molecules were better ranked than substrate, and then, also evaluated by the Molegro and Gold programs.Results: The HS23 and HS87 molecules were selected in common by the three programs, and ADME/Tox evaluation indicates they are not toxic. The molecular dynamics of PbHSD bonded to ligands showed stable complexes until 50 ns. To validate the results, compounds were purchased for assays of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), synergic profile with Amphotericin B (AmB) and cytotoxicity. The two molecules presented MIC of 32 µg/ml and MFC of 64 µg/ml against the P. brasiliensis (strain Pb18). They also showed synergistic activity with AmB and a lack of toxicity against Hela and Vero cell lines.Conclusion: These results suggest that the HS23 and HS87 are promising candidates as PbHSD inhibitors and may be used as hits for the development of new drugs against paracoccidioidomycosis.
[Box: see text].
Assuntos
Antifúngicos , Inibidores Enzimáticos , Homosserina Desidrogenase , Testes de Sensibilidade Microbiana , Paracoccidioides , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/enzimologia , Antifúngicos/farmacologia , Antifúngicos/química , Humanos , Homosserina Desidrogenase/antagonistas & inibidores , Homosserina Desidrogenase/metabolismo , Homosserina Desidrogenase/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Animais , Células Vero , Chlorocebus aethiops , Simulação de Acoplamento Molecular , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/microbiologia , Células HeLa , Brasil , Anfotericina B/farmacologia , Simulação de Dinâmica Molecular , Simulação por Computador , Sinergismo Farmacológico , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/químicaRESUMO
Acyl-homoserine lactones (AHLs) are quorum-sensing signaling molecules in Gram-negative bacteria and positively regulate biofilm formation in Salmonella under specific conditions. In this study, biofilm formation in Salmonella enterica was evaluated at 28 and 37 °C, under aerobic and anaerobic conditions. Additionally, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on biofilm formation and the expression of genes related to the synthesis of structural components, regulation, and quorum sensing was assessed under anaerobiosis at 28 and 37 °C. Biofilm formation was found not to be influenced by the atmospheric conditions at 28 °C. However, it was reduced at 37 °C under anaerobiosis. C12-HSL enhanced biofilm formation at 37 °C under anaerobiosis and increased the expression of the adrA and luxS genes, suggesting an increase in c-di-GMP, a second messenger that controls essential physiological functions in bacteria. These results provide new insights into the regulation of biofilm formation in Salmonella under anaerobic conditions.
Assuntos
Percepção de Quorum , Salmonella enteritidis , Percepção de Quorum/genética , Salmonella enteritidis/genética , Biofilmes , Anaerobiose , 4-Butirolactona/farmacologia , 4-Butirolactona/metabolismo , Acil-ButirolactonasRESUMO
Over the past decade, numerous publications have emerged in the literature focusing on the inhibition of quorum sensing (QS) by plant extracts and phenolic compounds. However, there is still a scarcity of studies that delve into the specific mechanisms by which these compounds inhibit QS. Thus, our question is whether phenolic compounds can inhibit QS in a specific or indirect manner and to elucidate the underlying mechanisms involved. This study is focused on the most studied QS system, namely, autoinducer type 1 (AI-1), represented by N-acyl-homoserine lactone (AHL) signals and the AHL-mediated QS responses. Here, we analyzed the recent literature in order to understand how phenolic compounds act at the cellular level, at sub-inhibitory concentrations, and evaluated by which QS inhibition mechanisms they may act. The biotechnological application of QS inhibitors holds promising prospects for the pharmaceutical and food industries, serving as adjunct therapies and in the prevention of biofilms on various surfaces.
RESUMO
Microfouling can have significant economic impacts for hydroelectric power plants. However, knowledge concerning the composition and metabolism of microbial biofilm in cooling systems remains scarce. We examined the metagenome present in a cooling system, comprising a filter (F) and heat exchanger (HE), in the Nova Ponte hydroelectric power plant in Brazil, to identify bacteria and pathways that could be targeted to monitor and control biofilm formation. Our data revealed that the microfouling sample from heat exchanger 1 (HEM1), with porous consistency, presented enriched bacterial members not frequently described as biofilm formers in cooling systems, besides it has been shown to be an autoinducer repression pathway. Furthermore, the microfouling sample from heat exchanger 2 (HEM2), with gelatinous consistency, seemed to be an established biofilm, containing enriched bacterial groups such as Desulfotomaculum and Crenothrix and autoinducers, with biotechnological relevance in industrial biofilms. The results demonstrate that biofilm composition will vary depending on different abiotic conditions and the antifouling strategy used, including type of compound, concentration, and frequency of use. Therefore, all these variables must be evaluated when a power plant is affected by microbial slime in the cooling system. Our findings could help to define strategies for efficient and ecofriendly measures to contain microfouling in power plants.
Assuntos
Incrustação Biológica , Incrustação Biológica/prevenção & controle , Biofilmes , Bactérias/genética , Brasil , Percepção de QuorumRESUMO
Bacteria can communicate to each other via quorum sensing, a cell density-dependent gene regulation system that stimulates the expression of virulence factors in the neighboring cells. Although the interaction of the natural product ajoene with the Hfq protein has been associated with the disruption of the quorum sensing system in Pseudomonas aeruginosa, there is no information concerning the corresponding ligand-target interaction process. Herein we observed a strong correlation (p < 0.00001) between the estimated affinities for the binding of 23 ajoene analogues at the proximal site of the Hfq protein of P. aeruginosa and their corresponding IC50 values, which reflect the reduction in the transcription of a virulence factor after quorum sensing inhibition. In this concern, our analyses reinforces previous propositions suggesting that ajoene could target the Hfq protein and affects its interaction with RNAs. Based on docking simulations, we tried to elucidate the binding mode of ajoene into the proximal Hfq site and we also established the minimum set of groups that would be necessary for a good interaction at this site, which includes a single hydrogen bond acceptor feature surrounded by groups that interact via π-sulfur (i.e., disulfide sulfurs) and/or π-alkyl/π-π stacking interactions (e.g., vinyl or small aryl/heteroaryl/heterocyclic groups). Because of the widespread role of Hfq as a matchmaker between messenger and small regulatory RNAs in Gram-negatives, we believe the discussion here provided for P. aeruginosa could be extrapolated for Gram-negatives in general, while the interaction of ajoene over the Hfq protein of Gram-positives would still remain more controversial.
Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Percepção de Quorum/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , RNA/metabolismo , Dissulfetos/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
The enzyme Homoserine dehydrogenase from Paracoccidioides brasiliensis (PbHSD), an interesting enzyme in the search for new antifungal drugs against paracoccidioidomycosis, was expressed by E. coli. Thirty milligrams of PbHSD with 94% of purity were obtained per liter of culture medium. The analysis by CD spectroscopy indicates a composition of 45.5 ± 7.3% of α-helices and 10.5 ± 7.0% ß-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectroscopy has shown stability of PbHSD in the presence of urea until Cm of 4.13 ± 0.21 M, and a broad pH range in which there is no conformational change. The protein analysis by differential scanning calorimetry indicates high stability at room temperature, but low stability at high temperatures, suffering irreversible denaturation, with Tm = 58.65 ± 0.87 °C. Kinetic studies of PbHSD by molecular absorption spectroscopy in UV/Vis have shown an optimum pH between 9.35 and 9.50, with Michaelian behavior, presenting KM of 224 ± 15 µM and specific activity at optimum pH of 2.10 ± 0.07 µmol/min/mg for homoserine. Therefore, protein expression and purification were efficient, and the structural characterization has shown that PbHSD presents native conformation with enzymatic activity in kinetic assays.
Assuntos
Paracoccidioides , Paracoccidioides/genética , Paracoccidioides/metabolismo , Homosserina Desidrogenase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Espectrometria de FluorescênciaRESUMO
Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, and one of the etiological agents of the disease is Paracoccidioides brasiliensis. Currently, available treatments present adversities, such as duration, side effects, and drug interactions. In search of new therapy possibilities, this study evaluates drugs approved for use against the homoserine dehydrogenase enzyme, by an in silico approach, which performs an important biosynthesis phase for the fungus and is not present in the human body. The three-dimensional structure of the homoserine dehydrogenase enzyme from Paracoccidioides brasiliensis was obtained by homology modeling. The model was validated, and simulations were performed for virtual screening of molecules of drugs approved from the Drugs-libs database by the MTiOpenScreen web server. Molecular dynamics in three replicas were used for four drugs with better results, and in two more molecules as a control, the HS9 with inhibition against enzyme and HON which shows inhibition against mold structure. Based on the results of molecular dynamics and the comparison of binding free energy, the drug that obtained the best result was Bemcentinib. In comparison with the controls, it presented a highly relevant affinity with - 44.63 kcal/mol, in addition to good structural stability and occupation of the active site. Therefore, Bemcentinib is a promising molecule for the inhibition of PbHSD protein (homoserine dehydrogenase of Paracoccidioides brasiliensis) and a therapeutic option to be investigated.
Assuntos
Paracoccidioides , Humanos , Paracoccidioides/metabolismo , Homosserina Desidrogenase , Reposicionamento de Medicamentos , Antifúngicos/farmacologiaRESUMO
Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, the most prevalent disease in salmonid species in Chilean salmonids farms. Many bacteria produce N-acyl-homoserine lactones (AHLs) as a quorum-sensing signal molecule to regulate gene expression in a cell density-dependent manner, and thus modulate physiological characteristics and several bacterial mechanisms. In this study, a fluorescent biosensor system method and gas chromatography-tandem mass spectrometry (GC/MS) were combined to detect AHLs produced by P. salmonis. These analyses revealed an emitted fluorescence signal when the biosensor P. putida EL106 (RPL4cep) was co-cultured with both, P. salmonis LF-89 type strain and an EM-90-like strain Ps007, respectively. Furthermore, the production of an AHL-type molecule was confirmed by GC/MS by both P. salmonis strains, which identified the presence of a N-acetyl-L-homoserine Lactone in the supernatant extract. However, It is suggested that an alternate pathway could synthesizes AHLs, which should be address in future experiments in order to elucidate this important bacterial process. To the best of our knowledge, the present report is the first to describe the type of AHLs produced by P. salmonis.
Assuntos
4-Butirolactona , Percepção de Quorum , 4-Butirolactona/análogos & derivados , Acil-Butirolactonas , Bactérias , Cromatografia Gasosa-Espectrometria de Massas , PiscirickettsiaRESUMO
Salmonella is a food and waterborne pathogen responsible for outbreaks worldwide, and it can survive during passage through the stomach and inside host phagocytic cells. Virulence genes are required for infection and survival in macrophages, and some are under the regulation of the quorum sensing (QS) system. This study investigated the influence of the autoinducer 1 (AI-1), N-dodecanoyl-homoserine lactone (C12-HSL), on the virulence of Salmonella PT4 using Galleria mellonella as an infection model. Salmonella PT4 was grown in the presence and absence of C12-HSL under anaerobic conditions for 7 h, and the expression of rpoS, arcA, arcB, and invA genes was evaluated. After the inoculation of G. mellonella with the median lethal dose (LD50) of Salmonella PT4, the survival of bacteria inside the larvae and their health status (health index scoring) were monitored, as well as the pigment, nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT) production. Also, the hemocyte viability, the induction of caspase-3, and microtubule-associated light chain 3 (LC3) protein in hemocytes were evaluated. Salmonella PT4 growing in the presence of C12-HSL showed increased rpoS, arcA, arcB, and invA expression and promoted higher larvae mortality and worse state of health after 24 h of infection. The C12-HSL also increased the persistence of Salmonella PT4 in the hemolymph and in the hemocytes. The highest pigmentation, NO production, and antioxidant enzymes were verified in the larva hemolymph infected with Salmonella PT4 grown with C12-HSL. Hemocytes from larvae infected with Salmonella PT4 grown with C12-HSL showed lower viability and higher production of caspase-3 and LC3. Taken together, these findings suggest that C12-HSL could be involved in the virulence of Salmonella PT4.
Assuntos
Homosserina , Salmonella enteritidis , 4-Butirolactona/análogos & derivados , Percepção de Quorum , Salmonella enteritidis/genética , VirulênciaRESUMO
It is known that plant and associated bacteria coevolved, but just now the roles of chemical signaling compounds in these intricate relationships have been systematically studied. Many Gram-negative bacteria produce N-acyl-L-homoserine lactones (AHL), chemical signals used in quorum-sensing bacterial communications mechanisms. In recent years, it has been shown that these compounds may also influence the development of plants, acting as allelochemicals, in still not well understood eukaryot-prokaryot interactions. In the present work, a quorum-sensing molecule produced by the tomato associated bacterium Pseudomonas sp. was characterized and its effects on germination and growth of tomato seedlings were accessed. The chemical study of the bacterium extract led to the identification of N-3-oxo-dodecanoyl-L-homoserine lactone (1), using gas chromatography coupled to electron impact mass spectrometry (GC-MS), and ultra-high resolution Qq-time-of-flight mass spectrometry (UHR-QqTOF-MS) equipped with an electrospray ionization source (ESI). The synthetic compound was tested at different concentrations in tomato to evaluate its effects on seed germination and seedlings root growth. Inhibition of tomato seed germination and root growth were observed in the presence of micromolar concentrations of the compound 1. Scanning electron microscopy evidenced morphological alterations on roots in the presence of the compound, with reduction of growth, impaired root hairs development and cracks in the rhizodermis.
Assuntos
Acil-Butirolactonas/metabolismo , Germinação/fisiologia , Pseudomonas/química , Solanum lycopersicum/fisiologia , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Raízes de Plantas/crescimento & desenvolvimentoRESUMO
Salmonella is a foodborne pathogen that can develop resistance to different stresses, which is essential for successful infection of the host. Some genes directly related to acid resistance are also involved in cationic peptide resistance in Gram-negative bacteria and could be under the control of quorum sensing (QS) mediated by autoinducer 1, known as acyl-homoserine lactone. Here, we investigated the influence of autoinducer 1, N-dodecanoyl-homoserine lactone (C12-HSL) on the resistance of Salmonella enterica subspecies enterica serovar Enteritidis to nisin and acid stress. Salmonella cells growing in anaerobic tryptic soy agar (TSB) at a pH of 7.0 for 7 h were submitted to acid stress at a pH of 4.5 in the presence and absence of nisin and were either supplemented or not with C12-HSL. Viable cell counts, gene expression, membrane charge alterations, fatty acid composition, and intracellular content leakage were observed. The autoinducer C12-HSL increased nisin resistance and survival at a pH of 4.5 in Salmonella. Also, C12-HSL increased the expression of the genes, phoP, phoQ, pmrA, and pmrB, which are involved with antimicrobial and acid resistance. The positive charge on the cell surface and concentration of cyclopropane fatty acid of the cellular membrane were increased in the presence of C12-HSL under acidic conditions, whereas membrane fluidity decreased. The loss of K+ and NADPH, promoted by nisin, was reduced in the presence of C12-HSL at a pH of 4.5. Taken together, these findings suggest that quorum sensing plays an important role in enhanced nisin and acid resistance in Salmonella.
Assuntos
Homosserina , Nisina , 4-Butirolactona/análogos & derivados , Nisina/farmacologia , Percepção de Quorum , Salmonella enteritidis/genéticaRESUMO
In the search of new and safe antibacterial compounds, the quorum sensing system (QS) modulation by natural products has been studied. As a result, many plant-derived compounds have been identified as potent quorum sensing inhibitors. Piper nigrum L. (black pepper) ethanolic extract inhibits the QS in some Gram-negative bacteria but the active components have not been previously identified. Thus, the objective of this work was to identify the P. nigrum peppercorns main components that block the QS, applying bioassay and chromatographic techniques. Piperine and trichostachine were identified as the main components responsible for the quorum quenching (QQ) activity of P. nigrum peppercorns extract. Piperine at 30 mg/L, decreased the violacein production by Chromobacterium violaceum CV026 by 35%, without affecting bacterial growth. Piperine concentration of 40 mg/L decreases violacein production by C. violaceum CV026 by 70% and growth in only 4.34%. Trichostachine at 50 mg/L decreases violacein production by C. violaceum CV026 by 12%, without affecting bacterial growth. P. nigrum extract concentration of 0.5 g/L decreased violacein production in 40 % and no effects on growth were observed. Neither P. nigrum extract, piperine, nor trichostachine did affect QS of Pseudomonas aeruginosa PAO1. Data here described exhibit the potential of piperamides as modulators of QS, not previously reported.
RESUMO
Quorum quenching (QQ), the obstruction of quorum sensing, is the most attractive way to break down the N-acyl-homoserine lactones (AHL) molecules. This work was focused at isolating AHL degrading bacteria from gastrointestinal tract of Oreochromis niloticus, with abilities appropriate for use as probiotic in aquaculture. The presence of an autoinducer inactivation (aiiA) homolog gene and AHL inactivation assay showed that Enterococcus faecium QQ12, which was one among the 20 isolates, could rapidly degrade synthetic C6-HSL in vitro and hampered violacein production by Chromobacterium violaceum. It had excellent biodegrading ability of natural N-AHL produced by Aeromonas hydrophila, suggesting that it can be used as a potential quencher bacterium for disrupting the virulence of A. hydrophila. It was susceptible to all the five antibiotics tried out. The isolate grew well at pH 3.0-7.0, was resistant to high level of bile salts (0-0.9%) and 0.5% of phenol. QQ12 also exhibited high degree of auto-aggregation and co-aggregation, confirming that it possessed good probiotic attributes. Goldfish fed diet incorporated with 108 and 1010 CFU g-1 of the QQ12 for 30 days showed 76.66-86.66% survival when challenged with A. hydrophila. The study indicates that Enterococcus faecium QQ12 could be used as a non-antibiotic feed additive in aquaculture to control bacterial diseases.
Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Ciclídeos/microbiologia , Enterococcus faecium/fisiologia , Doenças dos Peixes/microbiologia , Trato Gastrointestinal/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Probióticos/farmacologia , Percepção de Quorum , Aeromonas hydrophila/fisiologia , Animais , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Doenças dos Peixes/tratamento farmacológico , Carpa Dourada/microbiologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologiaRESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the genus Paracoccidioides spp., which mainly affects workers in rural regions of Latin America. Although the antifungal agents currently available for the treatment of PCM are effective in controlling the disease, many months are needed for healing, making the side effects and drug interactions relevant. In addition, conventional treatments are not able to control the sequelae left by PCM, even after the cure, justifying the search for new therapeutic options against PCM. In this context, the enzyme homoserine dehydrogenase of P. brasiliensis (PbHSD) was used to screen a library of natural products from the Zinc database using three different docking programs, i.e. Autodock, Molegro, and CLC Drugdiscovery Workbench. Three molecules (Zinc codes 2123137, 15967722, and 20611644) were better ranked than the homoserine substrate (HSE) and were used for in vitro trials of the minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MCF). All three molecules presented a fungicidal profile with MICs/MCFs of 8, 32, and 128 µg mL-1, respectively. The two most promising molecules presented satisfactory results with wide therapeutic ranges in the cytotoxicity assays. Molecular dynamics simulations of PbHSD indicated that the ligands remained bound to the protein by a common mechanism throughout the simulation. The molecule with the lowest MIC value presented the highest number of contacts with the protein. The results presented in this work suggest that the molecule Zinc2123137 may be considered as a hit in the development of new therapeutic options for PCM.
Assuntos
Antifúngicos/farmacologia , Homosserina Desidrogenase/antagonistas & inibidores , Paracoccidioides/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HeLa , Humanos , Ligantes , Testes de Sensibilidade Microbiana/métodos , Simulação de Dinâmica Molecular , Células VeroRESUMO
Bacteria rely on chemical communication to sense the environment and to retrieve information on their population densities. Accordingly, a vast repertoire of molecules is released, which synchronizes expression of genes, coordinates behavior through a process termed quorum-sensing (QS), and determines the relationships with eukaryotic species. Already identified QS molecules from Gram negative bacteria can be grouped into two main classes, N-acyl-L-homoserine lactones (AHLs) and cyclodipeptides (CDPs), with roles in biofilm formation, bacterial virulence or symbiotic interactions. Noteworthy, plants detect each of these molecules, change their own gene expression programs, re-configurate root architecture, and activate defense responses, improving in this manner their adaptation to natural and agricultural ecosystems. AHLs may act as alarm signals, pathogen and/or microbe-associated molecular patterns, whereas CDPs function as hormonal mimics for plants via their putative interactions with the auxin receptor Transport Inhibitor Response1 (TIR1). A major challenge is to identify the molecular pathways of QS-mediated crosstalk and the plant receptors and interacting proteins for AHLs, CDPs and related signals.
Assuntos
Raízes de Plantas/microbiologia , Percepção de Quorum/fisiologia , Rhizobiaceae/metabolismo , Interações Hospedeiro-Patógeno , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/fisiologiaRESUMO
AIM: Novel 4-methoxy-naphthalene derivatives were synthesized based on hits structures in order to evaluate the antifungal activity against Paracoccidioides spp. METHODS: Antifungal activity of compounds was evaluated against P. brasiliensis and most promising compounds 2 and 3 were tested against eight clinically important fungal species. RESULTS: Compound 3 was the more active compound with MIC 8 to 32 µg.ml-1 for Paracoccidioides spp without toxicity monkey kidney and murine macrophagecells. Carbohydrazide 3 showed good synergistic antifungal activity with amphotericin B against P. brasiliensis specie. Titration assay of carbohydrazide 3 with PbHSD enzyme demonstrates the binding ligand-protein. Molecular dynamics simulations show that ligand 3 let the PbHSD protein more stable. CONCLUSION: New carbohydrazide 3 is an attractive lead for drug development to treat paracoccidioidomycoses.
Assuntos
Antifúngicos/farmacologia , Naftalenos/farmacologia , Paracoccidioides/efeitos dos fármacos , Paracoccidioidomicose/tratamento farmacológico , Anfotericina B/farmacologia , Animais , Antifúngicos/uso terapêutico , Chlorocebus aethiops , Combinação de Medicamentos , Sinergismo Farmacológico , Homosserina Desidrogenase/metabolismo , Hidrazinas/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Naftalenos/síntese química , Naftalenos/uso terapêutico , Paracoccidioides/patogenicidade , Estabilidade Proteica , Células Vero/efeitos dos fármacosRESUMO
Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E.coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.
RESUMO
Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.
Assuntos
Azospirillum brasilense/fisiologia , Percepção de Quorum , Acil-Butirolactonas/metabolismo , Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Brasil , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Raízes de Plantas/microbiologia , Plasmídeos/genética , Plasmídeos/metabolismo , Zea mays/microbiologiaRESUMO
Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E. coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.