RESUMO
Antibiotic-resistant superbug bacteria represent a global health problem with no imminent solutions. Here we demonstrate that the combination (termed AB569) of acidified nitrite (A-NO2-) and Na2-EDTA (disodium ethylenediaminetetraacetic acid) inhibited all Gram-negative and Gram-positive bacteria tested. AB569 was also efficacious at killing the model organism Pseudomonas aeruginosa in biofilms and in a murine chronic lung infection model. AB569 was not toxic to human cell lines at bactericidal concentrations using a basic viability assay. RNA-Seq analyses upon treatment of P. aeruginosa with AB569 revealed a catastrophic loss of the ability to support core pathways encompassing DNA, RNA, protein, ATP biosynthesis, and iron metabolism. Electrochemical analyses elucidated that AB569 produced more stable SNO proteins, potentially explaining one mechanism of bacterial killing. Our data implicate that AB569 is a safe and effective means to kill pathogenic bacteria, suggesting that simple strategies could be applied with highly advantageous therapeutic/toxicity index ratios to pathogens associated with a myriad of periepithelial infections and related disease scenarios.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Ácido Edético/farmacologia , Nitrito de Sódio/farmacologia , Animais , Antibacterianos/uso terapêutico , Biofilmes/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Farmacorresistência Bacteriana/efeitos dos fármacos , Ácido Edético/química , Pneumopatias/tratamento farmacológico , Pneumopatias/microbiologia , Redes e Vias Metabólicas , Camundongos , Nitritos/química , Nitritos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
The presence of cariogenic bacteria within the prepared tooth cavity at the adhesive resin-dentin interface is detrimental to the long-term stability and function of composite restorations. Here, we report the synthesis and incorporation of methacrylated azobenzene nanogels within bisphenol A-glycidyl methacrylate/hydroxyethyl methacrylate/ethanol (B/H/E) adhesive resins and evaluate their ability to reduce the bacterial invasion of cariogenic Streptococcus mutans biofilms while preserving the mechanical strength and structural integrity of the critical interfacial connection between the restoration and the tooth. The azobenzene nanogel, with a hydrodynamic radius of < 2 nm and a molecular weight of 12,000 Da, was polymerized within B/H/E adhesive formulations at concentrations of 0.5 wt.%, 1.5 wt.%, and 2.5 wt.%. While the double-bond conversion, cytocompatibility, water solubility, and sorption of the adhesive networks were comparable, azobenzene nanogel networks showed improved hydrophobicity with a ≥ 25° increase in water contact angle. The polymerized adhesive surfaces formulated with azobenzene nanogels showed a 66% reduction in bacterial biofilms relative to the control while maintaining the mechanical properties and micro-tensile bond strength of the adhesive networks. The increased hydrophobicity and antibacterial activity are promising indicators that azobenzene nanogel additives have the potential to increase the durability and longevity of adhesive resins.
Assuntos
Resinas Compostas , Colagem Dentária , Antibacterianos/farmacologia , Compostos Azo , Resinas Compostas/química , Cimentos Dentários , Dentina/química , Adesivos Dentinários/química , Odontologia , Teste de Materiais , Metacrilatos/química , Nanogéis , Resistência à TraçãoRESUMO
Compare the effectiveness of selected dental lasers for decontamination of machined titanium surfaces in vitro. Seventy-two sterile machined surface titanium discs were individually inoculated with strains of Streptococcus mutans (Sm), Streptococcus oralis (So), Aggregatibacter actinomycetemcomitans (Aa), or all three bacteria together (MIX) at 34.0° C, 20.8% O2 and 5% CO2 for 12 h. After incubation, the discs were divided into six groups: 1) no treatment, 2) 0.12% chlorhexidine gluconate (CHX), and 3) 10,600 CO2, 4) 810 nm diode, 5) 2780 nm Er,Cr:YSGG, 6) 1064 nm Nd:YAG laser groups. After treatment, any remaining viable bacteria were liberated from the discs via sonication, transferred onto brain heart infusion (BHI) agar plates for culturing, and colony-forming units (CFUs) were recorded. Statistical analysis was performed. There were statistically significantly differences (SSD) (p < 0.01) in bacterial reduction of discs individually inoculated with Aa between the Er,Cr:YSGG and Nd:YAG lasers. There was also a SSD (p < 0.01) lower effect with the MIX with the Er,Cr:YSGG compared with all other modalities. Bacterial reduction with the CO2 was better (p < 0.001) than treatment with CHX or the Er,Cr:YSGG laser on killing of So. Although all modalities of treatment showed a mean of 98% or greater viable bacterial reduction, the most consistent bacterial reduction of all titanium discs was with the Nd:YAG laser (100%).
Assuntos
Lasers de Estado Sólido , Titânio , Aggregatibacter actinomycetemcomitans , Biofilmes , Lasers de Estado Sólido/uso terapêutico , Propriedades de SuperfícieRESUMO
In this issue of Journal of Bacteriology, Price et al. show that the Pseudomonas aeruginosa-produced exopolysaccharide alginate protects Staphylococcus aureus by dampening the expression of P. aeruginosa virulence products that usually inhibit S. aureus respiration and cell membrane integrity when the two organisms compete in other environments (C. E. Price, D. G. Brown, D. H. Limoli, V. V. Phelan, and G. A. O'Toole, J Bacteriol 202:e00559-19, 2020, https://doi.org/10.1128/jb.00559-19). This is the first report that exogenously added alginate affects P. aeruginosa competition and provides a partial explanation for S. aureus and P. aeruginosa coinfections in cystic fibrosis.
Assuntos
Alginatos/metabolismo , Fibrose Cística/microbiologia , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/metabolismo , Alginatos/química , Coinfecção/microbiologia , Humanos , Interações Microbianas , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genéticaRESUMO
Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.
Assuntos
Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/genética , Drosophila/genética , Camundongos , Mutação , Transcriptoma , Fatores de Virulência/genéticaRESUMO
Due to the prevalence of biofilm-related infections, which are mediated by bacterial quorum sensing, there is a critical need for materials and coatings that resist biofilm formation. We have developed novel anti-biofilm coatings that disrupt quorum sensing in surface-associated bacteria via the immobilization of acylase in polyurethane films. Specifically, acylase from Aspergillus melleus was covalently immobilized in biomedical grade polyurethane coatings via multipoint covalent immobilization. Coatings containing acylase were enzymatically active and catalyzed the hydrolysis of the quorum sensing (QS) molecules N-butyryl-L-homoserine lactone (C4-LHL), N-hexanoyl-L-homoserine lactone (C6-LHL), and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-LHL). In biofilm inhibition assays, immobilization of acylase led to an approximately 60% reduction in biofilm formation by Pseudomonas aeruginosa ATCC 10145 and PAO1. Inhibition of biofilm formation was consistent with a reduction in the secretion of pyocyanin, indicating the disruption of quorum sensing as the mechanism of the coating activity. Scanning electron microscopy further showed that acylase-containing coatings contained far fewer bacterial cells than control coatings that lacked acylase. Moreover, acylase-containing coatings retained 90% activity when stored dry at 37°C for 7 days and were more stable than the free enzyme in physiological conditions, including artificial urine. Ultimately, such coatings hold considerable promise for the clinical management of catheter-related infections as well as the prevention of infections in orthopedic applications (i.e., on hip and knee prostheses) and on contact lenses. Biotechnol. Bioeng. 2016;113: 2535-2543. © 2016 Wiley Periodicals, Inc.
Assuntos
Amidoidrolases/administração & dosagem , Antibacterianos/administração & dosagem , Aspergillus/enzimologia , Biofilmes/crescimento & desenvolvimento , Poliuretanos/química , Pseudomonas aeruginosa/fisiologia , Amidoidrolases/química , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/administração & dosagem , Materiais Revestidos Biocompatíveis/síntese química , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/fisiologiaRESUMO
Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RT-qPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ΔalgZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Transativadores/metabolismo , Alginatos/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transativadores/genética , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
The transcriptional regulator BrlR is a member of the MerR family of multidrug transport activators that contributes to the high-level drug tolerance of Pseudomonas aeruginosa biofilms. While MerR regulators are known to activate both the expression of multidrug efflux pump genes and their own transcription upon inducer binding, little is known about BrlR activation. We demonstrate using promoter reporter strains, in vivo and in vitroâ DNA-binding assays combined with 5'RACE, that BrlR binds to its own promoter, likely via a MerR-like palindromic sequence. Unlike known MerR multidrug transport activators, BrlR and brlR expression are not activated by multidrug transporter substrates. Instead, BrlR-DNA binding was enhanced by the secondary messenger c-di-GMP. In addition to enhanced BrlR-DNA binding, c-di-GMP levels contributed to PbrlR promoter activity in initial attached cells with elevated c-di-GMP levels correlating with increased expression of brlR. While not harbouring amino acid motifs resembling previously defined c-di-GMP-binding domains, BrlR was found to bind c-di-GMPâ in vitro at a ratio of one c-di-GMP per two BrlR. Cross-linking assays confirmed dimer formation to be enhanced in the presence of elevated c-di-GMP levels. Our findings demonstrate BrlR to be an unusual MerR-family member in that BrlR function and expression require the secondary messenger c-di-GMP.
Assuntos
GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismo , GMP Cíclico/metabolismo , DNA Bacteriano/metabolismo , Genes Reporter , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização ProteicaRESUMO
Chronic nonhealing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building on prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the three-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing.
Assuntos
Anti-Infecciosos Locais/farmacologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Lesões dos Tecidos Moles/patologia , Triptofano/farmacologia , Cicatrização , Infecção dos Ferimentos/patologia , Animais , Bandagens , Biofilmes/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Infecções por Pseudomonas/microbiologia , Lesões dos Tecidos Moles/microbiologia , Infecção dos Ferimentos/microbiologiaRESUMO
Accurate and rapid assessment of the healing status of a wound in a simple and noninvasive manner would enable clinicians to diagnose wounds in real time and promptly adjust treatments to hasten the resolution of nonhealing wounds. Histologic and biochemical characterization of biopsied wound tissue, which is currently the only reliable method for wound assessment, is invasive, complex to interpret, and slow. Here we demonstrate the use of Raman microspectroscopy coupled with multivariate spectral analysis as a simple, noninvasive method to biochemically characterize healing wounds in mice and to accurately identify different phases of healing of wounds at different time-points. Raman spectra were collected from "splinted" full thickness dermal wounds in mice at 4 time-points (0, 1, 5, and 7 days) corresponding to different phases of wound healing, as verified by histopathology. Spectra were deconvolved using multivariate factor analysis (MFA) into 3 "factor score spectra" (that act as spectral signatures for different stages of healing) that were successfully correlated with spectra of prominent pure wound bed constituents (i.e., collagen, lipids, fibrin, fibronectin, etc.) using non-negative least squares (NNLS) fitting. We show that the factor loadings (weights) of spectra that belonged to wounds at different time-points provide a quantitative measure of wound healing progress in terms of key parameters such as inflammation and granulation. Wounds at similar stages of healing were characterized by clusters of loading values and slowly healing wounds among them were successfully identified as "outliers". Overall, our results demonstrate that Raman spectroscopy can be used as a noninvasive technique to provide insight into the status of normally healing and slow-to-heal wounds and that it may find use as a complementary tool for real-time, in situ biochemical characterization in wound healing studies and clinical diagnosis.
Assuntos
Análise Espectral Raman/métodos , Cicatrização/fisiologia , Ferimentos e Lesões/classificação , Ferimentos e Lesões/patologia , Animais , Biópsia , Análise Fatorial , Tecido de Granulação/química , Inflamação/metabolismo , Análise dos Mínimos Quadrados , Camundongos , Camundongos Endogâmicos BALB C , Análise Multivariada , Pele/químicaRESUMO
Corynebacterium bovis infection in biomedical research is synonymous with skin hyperkeratosis of athymic nude mice. This clinical sign can be obvious and is the namesake for 'scaly skin disease.' Other clinical signs that accompany scaly skin, including early presentation, duration, and rate of resolution, are less well known. The goal of this study was to characterize the clinical signs of C. bovis infection in nude mice under experimental conditions and develop a quantifiable scoring system. For the development, prospective trial, and application of this clinical scoring system, 93 naïve Hsd:Athymic Nude mice were used, of which 81 were exposed to soiled bedding from clinically ill C. bovis-infected NSG mice. The emergence of clinical signs was monitored and scored daily for 14 d. We identified 3 categories of clinical signs including skin hyperemia, skin hyperkeratosis, and surrogate indicators of overall health. Each of these defined categories appeared consistently and progressed and regressed temporally. We subsequently used this scoring system to determine if the age of Hsd:Athymic Nude mice (6 compared with 10 wk) at time of infection affects clinical severity. Our findings demonstrate that 6-wk-old mice demonstrate more severe clinical signs. Ten-week-old mice showed less skin hyperemia and no skin hyperkeratosis and were less affected by the infection based on surrogates of overall health. Here we show the utility of this novel scoring system and the impact of nude mouse age at the time of infection on C. bovis clinical disease.
RESUMO
A common concern in preclinical cancer research is the introduction of Corynebacterium bovis into immunodeficient mouse colonies through cancer cell lines. C. bovis is a known contaminant of patient-derived xenograft tumors passaged horizontally between immunodeficient mice. However, it is unclear if C. bovis can grow in mammalian tissue culture conditions or tissue culture media. We hypothesized that C. bovis would not grow under tissue culture conditions or media, diminishing the risk of transmission from tumor cell lines cultured in vitro. Three C. bovis isolates, CUAMC1, HAC, and ATCC-7715, were used to test our hypothesis in 3 of the most common media used to grow human cancer cell lines including RPMI 1640 + 10% FBS (RPMI), DMEM/high glucose + 10% FBS (DMEM), and DMEM/F-12 + 10% FBS (DMEM/F12). Our results confirmed propagation of each C. bovis isolate in DMEM/F12 media under tissue culture conditions after 72 h. However, these results also demonstrate diminished viability of each C. bovis isolate in RPMI and DMEM after 72 h. To assess whether antibiotics could halt the growth of C. bovis under tissue culture conditions in DMEM/F12, penicillin-streptomycin (pen/strep) was added to the experimental media. This treatment was effective in eliminating all viable C. bovis in the culture system after 72 h. Our data suggest that C. bovis growth under tissue culture conditions is possible and growth in tissue culture media is nuanced. These results highlight the importance of pathogen surveillance for tumor cell lines propagated in vitro and demonstrate the need for further investigation into C. bovis growth requirements.
RESUMO
A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , DNA Bacteriano/metabolismo , Tolerância a Medicamentos , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Immunoblotting , Testes de Sensibilidade Microbiana , Ligação Proteica , Pseudomonas aeruginosa/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response, resulting in a colony morphology and phenotype referred to as mucoid. However, how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, by transcriptomics, and in a murine acute virulence model. The PA14 nonredundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan remodeling, uptake of phosphate and iron, phenazine biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa cultures growing in the presence of vanadate showed differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa, but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study.
Assuntos
Alginatos/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Vanadatos/farmacologia , Doença Aguda , Ágar , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Meios de Cultura/química , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Vanadatos/metabolismo , Virulência/genéticaRESUMO
AlgR is a key Pseudomonas aeruginosa transcriptional response regulator required for virulence. AlgR activates alginate production and twitching motility but represses the Rhl quorum-sensing (QS) system, including rhamnolipid production. The role of AlgR phosphorylation is enigmatic, since phosphorylated AlgR (AlgR-P) is required for twitching motility through the fimU promoter but is not required for the activation of alginate production. In order to examine the role of AlgR phosphorylation in vivo, a PAO1 algRD54E strain (with algR encoding a D-to-E change at position 54), which constitutively activates fimU transcription and exhibits twitching motility, was created. A corresponding PAO1 algRD54N strain (with algR encoding a D-to-N change at position 54) that does not activate fimU or twitching motility was compared to PAO1, PAO1 algRD54E, PAO1 ΔalgZ (deletion of the algZ [fimS] gene, encoding a putative histidine kinase), and PAO1 ΔalgR for swarming motility, rhamnolipid production, and rhlA transcription. PAO1 and PAO1 algRD54E produced approximately 2-fold-higher levels of rhamnolipids than PAO1 algRD54N and PAO1 ΔalgZ, thereby indicating that phosphorylated AlgR is required for normal rhamnolipid production. Examination of purified AlgR, AlgR-P, AlgR D54N, and AlgR D54E showed that AlgR-P and AlgR D54E bound preferentially to the fimU and rhlA promoters. Additionally, AlgR-P bound specifically to two sites within the rhlA promoter that were not bound by unphosphorylated AlgR. Taken together, these results indicate that phosphorylated AlgR-P has increased affinity for the rhlA promoter and is required for the coordinate activation of twitching motility, rhamnolipid production, and swarming motility in P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/metabolismo , Locomoção , Pseudomonas aeruginosa/fisiologia , Transativadores/metabolismo , Transcrição Gênica , Proteínas de Bactérias/genética , Proteínas de Fímbrias/biossíntese , Deleção de Genes , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Fosforilação , Pseudomonas aeruginosa/genética , Transativadores/genéticaRESUMO
A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that microcolony formation is associated with stressful, oxygen-limiting but electron-rich conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation, while addition of pyruvate partly restored microcolony formation in mifR mutant biofilms. In contrast, expression of ldhA in mifR::Mar fully restored microcolony formation by this mutant strain. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation of the P. aeruginosa biofilm environment.
Assuntos
Biofilmes/crescimento & desenvolvimento , Redes e Vias Metabólicas/genética , Pseudomonas aeruginosa/fisiologia , Ácido Pirúvico/metabolismo , Meios de Cultura/química , Fermentação , Deleção de Genes , Perfilação da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismoRESUMO
Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the d and l isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of d and l isoforms producing the greatest inhibitory effect. Addition of d-/l-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa.
Assuntos
Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Triptofano/farmacologiaRESUMO
BACKGROUND: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ(22)) to initiate transcription of the alginate biosynthetic operon. RESULTS: In the current study, we mapped the mucE transcriptional start site, and determined that P(mucE) activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P(mucE) activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. CONCLUSIONS: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Peptídeo Hidrolases/biossíntese , Pseudomonas aeruginosa/genética , Fator sigma/metabolismo , Transcrição Gênica , Alginatos , Ácido Glucurônico/biossíntese , Ácidos Hexurônicos , Regiões Promotoras Genéticas , Sítio de Iniciação de TranscriçãoRESUMO
Pseudomonas aeruginosa and Staphylococcus aureus are among the most frequently isolated bacterial species from polymicrobial infections of patients with cystic fibrosis and chronic wounds. We apply mass spectrometry guided interaction studies to determine how chemical interaction shapes the fitness and community structure during co-infection of these two pathogens. We demonstrate that S. aureus is equipped with an elegant mechanism to inactivate pyochelin via the yet uncharacterized methyltransferase Spm (staphylococcal pyochelin methyltransferase). Methylation of pyochelin abolishes the siderophore activity of pyochelin and significantly lowers pyochelin-mediated intracellular reactive oxygen species (ROS) production in S. aureus. In a murine wound co-infection model, an S. aureus mutant unable to methylate pyochelin shows significantly lower fitness compared with its parental strain. Thus, Spm-mediated pyochelin methylation is a mechanism to increase S. aureus survival during in vivo competition with P. aeruginosa.