RESUMEN
Saccharomyces cerevisiae has long been used as a model organism to study genome instability. The SAM1 and SAM2 genes encode AdoMet synthetases, which generate S-AdenosylMethionine (AdoMet) from Methionine (Met) and ATP. Previous work from our group has shown that deletions of the SAM1 and SAM2 genes cause changes to AdoMet levels and impact genome instability in opposite manners. AdoMet is a key product of methionine metabolism and the major methyl donor for methylation events of proteins, RNAs, small molecules, and lipids. The methyl cycle is interrelated to the folate cycle which is involved in de novo synthesis of purine and pyrimidine deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP). AdoMet also plays a role in polyamine production, essential for cell growth and used in detoxification of reactive oxygen species (ROS) and maintenance of the redox status in cells. This is also impacted by the methyl cycle's role in production of glutathione, another ROS scavenger and cellular protectant. We show here that sam2∆/sam2∆ cells, previously characterized with lower levels of AdoMet and higher genome instability, have a higher level of each dNTP (except dTTP), contributing to a higher overall dNTP pool level when compared to wildtype. Unchecked, these increased levels can lead to multiple types of DNA damage which could account for the genome instability increases in these cells.
Asunto(s)
S-Adenosilmetionina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Inestabilidad Genómica , Desoxirribonucleótidos/metabolismo , Nucleótidos/metabolismo , Metionina/metabolismoRESUMEN
Cutaneous thermal injuries from burns/explosives are a major cause of morbidity and mortality and represent a monumental burden on our current health care system. Injury severity is predominantly due to potentially lethal sepsis caused by multidrug-resistant (MDR) bacteria such as Pseudomonas aeruginosa (MDR-PA). Thus, there is a critical need to develop novel and effective antimicrobials for the (i) prevention, (ii) treatment, and (iii) healing of such wounds that are complicated by MDR-P. aeruginosa and other bacterial infections. AB569 is a novel bactericidal tandem consisting of acidified NaNO2 (A-NO2-) and Na2-EDTA. Here, we first show that AB569 acts synergistically to kill all human burn wound strains of P. aeruginosa in vitro. This was found to be due, in part, to the generation of A-NO2--mediated nitric oxide (NO) formation coupled with the metal chelating properties of Na2-EDTA. Using a murine scald burn wound model of P. aeruginosa infection, an AB569-Solosite gel formulation eradicated all bacteria. Futher, we also demonstrate enhanced AB569-mediated wound healing by not only accelerating wound contraction, but also by reducing levels of the proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß while increasing the levels of anti-inflammatory cytokine, IL-10, and granulocyte-colony-stimulating factor (G-CSF). We also observed better epidermal restoration in AB569-treated wounds. Taken together, we conclude that this study provides solid foundational evidence that AB569 can be used topically to treat highly problematic dermal insults, including wound, burn, blast, and likely, diabetic infections in civilian and military populations, and help relieve the economical burden that MDR organisms have on the global health care system.
Asunto(s)
Antibacterianos/farmacología , Quemaduras/tratamiento farmacológico , Ácido Edético/farmacología , Nitritos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Quemaduras/complicaciones , Quemaduras/microbiología , Colágeno/genética , Modelos Animales de Enfermedad , Ácido Edético/uso terapéutico , Geles , Óxido Nítrico/biosíntesis , Nitritos/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológicoRESUMEN
Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Campaña Afgana 2001- , Antibacterianos/farmacología , Desinfectantes/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Ácido Edético/farmacología , Guerra de Irak 2003-2011 , Nitritos/farmacología , Infección de Heridas/microbiología , Infecciones por Acinetobacter/epidemiología , Acinetobacter baumannii/genética , Adulto , Afganistán/epidemiología , Antibacterianos/química , Biopelículas/efectos de los fármacos , Células Cultivadas , Desinfectantes/química , Combinación de Medicamentos , Farmacorresistencia Bacteriana Múltiple/genética , Ácido Edético/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Irak/epidemiología , Pruebas de Sensibilidad Microbiana , Nitritos/química , Reacción en Cadena de la Polimerasa , Piel/citología , Infección de Heridas/epidemiologíaRESUMEN
Microbial extracellular electron transfer (EET) stimulates a plethora of intellectual concepts leading to potential applications that offer environmentally sustainable advances in the fields of biofuels, wastewater treatment, bioremediation, desalination, and biosensing. Despite its vast potential and remarkable research efforts to date, bacterial electrogenicity is arguably the most underdeveloped technology used to confront the aforementioned challenges. Severe limitations are placed in the intrinsic energy and electron transfer processes of naturally occurring microorganisms. Significant boosts in this technology can be achieved with the growth of synthetic biology tools that manipulate microbial electron transfer pathways and improve their electrogenic potential. In particular, electrogenic Pseudomonas aeruginosa has been studied with the utility of its complete genome being sequenced coupled with well-established techniques for genetic manipulation. To optimize power density production, a high-throughput, rapid and highly sensitive test array for measuring the electrogenicity of hundreds of genetically engineered P. aeruginosa mutants is needed. This task is not trivial, as the accurate and parallel quantitative measurements of bacterial electrogenicity require long measurement times (~tens of days), continuous introduction of organic fuels (~tends of milliliters), architecturally complex and often inefficient devices, and labor-intensive operation. The overall objective of this work was to enable rapid (<30 min), sensitive (>100-fold improvement), and high-throughput (>96 wells) characterization of bacterial electrogenicity from a single 5 µL culture suspension. This project used paper as a substratum that inherently produces favorable conditions for easy, rapid, and sensitive control of an electrogenic microbial suspension. From 95 isogenic P. aeruginosa mutant, an hmgA mutant generated the highest power density (39 µW/cm2), which is higher than that of wild-type P. aeruginosa and even the strongly electrogenic organism, Shewanella oneidensis (25 µW/cm2). In summary, this work will serve as a springboard for the development of novel paradigms for genetic networks that will help develop mutations or over-expression and synthetic biology constructs to identify genes in P. aeruginosa and other organisms that enhance electrogenic performance in microbial fuel cells (MFCs).
Asunto(s)
Fuentes de Energía Bioeléctrica/microbiología , Pseudomonas aeruginosa/metabolismo , Electricidad , Transporte de Electrón , Electrones , Diseño de Equipo , Ingeniería Genética , Genoma Bacteriano , Mutación , Pseudomonas aeruginosa/genéticaRESUMEN
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.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Ácido Edético/farmacología , Nitrito de Sodio/farmacología , Animales , Antibacterianos/uso terapéutico , Biopelículas/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo , Farmacorresistencia Bacteriana/efectos de los fármacos , Ácido Edético/química , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/microbiología , Redes y Vías Metabólicas , Ratones , Nitritos/química , Nitritos/farmacología , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157-194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases.
Asunto(s)
Proteínas Bacterianas/genética , Biopelículas , Pulmón/microbiología , Mutación , Nitritos/farmacología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Enfermedad Crónica , Humanos , Concentración de Iones de Hidrógeno , Plancton/metabolismo , Plancton/fisiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
The global pandemic of antibiotic resistance is an ever-burgeoning public health challenge, motivating the development of adjunct bactericidal therapies. Nitric oxide (NO) is a potent bioactive gas that induces a variety of therapeutic effects, including bactericidal and biofilm dispersion properties. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to characterize NO-loaded microbubbles (MB) stabilized with a lipid shell and to assess the feasibility of antibacterial therapy in vitro. MB were loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) (9:1 v/v and 1:1 v/v). The size distribution and acoustic attenuation coefficient of NO-MB and NO-OFP-MB were measured. Ultrasound-triggered release of the encapsulated gas payload was demonstrated with 3-MHz pulsed Doppler ultrasound. An amperometric microelectrode sensor was used to measure NO concentration released from the MB and compared to an NO-OFP-saturated solution. The effect of NO delivery on the viability of planktonic (free living) Staphylococcus aureus (SA) USA 300, a methicillin-resistant strain, was evaluated in a 96 well-plate format. The co-encapsulation of NO with OFP increased the total volume and attenuation coefficient of MB. The NO-OFP-MB were destroyed with a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure (in situ MI of 1.34) but maintained their echogenicity when exposed to 0.02 MPa peak negative pressure (in situ MI of 0.01. The NO dose in NO-MB and NO-OFP-MB was more than 2-fold higher than the NO-OFP-saturated solution. Delivery of NO-OFP-MB increased bactericidal efficacy compared to the NO-OFP-saturated solution or air and OFP-loaded MB. These results suggest that encapsulation of NO with OFP in lipid-shelled MB enhances payload delivery. Furthermore, these studies demonstrate the feasibility and limitations of NO-OFP-MB for antibacterial applications.
RESUMEN
Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are chronic pulmonary diseases that affect ~70,000 and 251 million individuals worldwide, respectively. Although these two diseases have distinctly different pathophysiologies, both cause chronic respiratory insufficiency that erodes quality of life and causes significant morbidity and eventually death. In both CF and COPD, the respiratory microbiome plays a major contributing role in disease progression and morbidity. Pulmonary pathogens can differ dramatically during various stages of each disease and frequently cause acute worsening of lung function due to disease exacerbation. Despite some similarities, outcome and timing/type of exacerbation can also be quite different between CF and COPD. Given these clinical distinctions, both patients and physicians should be aware of emerging therapeutic options currently being offered or in development for the treatment of lung infections in individuals with CF and COPD. Although interventions are available that prolong life and mitigate morbidity, neither disorder is curable. Both acute and chronic pulmonary infections contribute to an inexorable downward course and may trigger exacerbations, culminating in loss of lung function or respiratory failure. Knowledge of the pulmonary pathogens causing these infections, their clinical presentation, consequences, and management are, therefore, critical. In this review, we compare and contrast CF and COPD, including underlying causes, general outcomes, features of the lung microbiome, and potential treatment strategies.
RESUMEN
Pseudomonas aeruginosa is a metabolically voracious bacterium that is easily manipulated genetically. We have previously shown that the organism is also highly electrogenic in microbial fuel cells (MFCs). Polarization studies were performed in MFCs with wild-type strain PAO1 and three mutant strains (pilT, bdlA and pilT bdlA). The pilT mutant was hyperpiliated, while the bdlA mutant was suppressed in biofilm dispersion chemotaxis. The double pilT bdlA mutant was expected to have properties of both mutations. Polarization data indicate that the pilT mutant showed 5.0- and 3.2-fold increases in peak power compared to the wild type and the pilT bdlA mutant, respectively. The performance of the bdlA mutant was surprisingly the lowest, while the pilT bdlA electrogenic performance fell between the pilT mutant and wild-type bacteria. Measurements of biofilm thickness and bacterial viability showed equal viability among the different strains. The thickness of the bdlA mutant, however, was twice that of wild-type strain PAO1. This observation implicates the presence of dead or dormant bacteria in the bdlA mutant MFCs, which increases biofilm internal resistance as confirmed by electrochemical measurements.
Asunto(s)
Fuentes de Energía Bioeléctrica , Biopelículas , Pseudomonas aeruginosa/genética , MutaciónRESUMEN
The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , terc-Butilhidroperóxido/farmacología , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Pseudomonas aeruginosa (PA) is an important airway pathogen of cystic fibrosis and chronic obstructive disease patients. Multiply drug resistant PA is becoming increasing prevalent and new strategies are needed to combat such insidious organisms. We have previously shown that a mucoid, mucA22 mutant PA is exquisitely sensitive to acidified nitrite ([Formula: see text], pH 6.5) at concentrations that are well tolerated in humans. Here, we used a transposon mutagenesis approach to identify PA mutants that are hypersensitive to [Formula: see text]. Among greater than 10,000 mutants screened, we focused on PA4455, in which the transposon was found to disrupt the production of a putative cytoplasmic membrane-spanning ABC transporter permease. The PA4455 mutant was not only highly sensitive to [Formula: see text], but also the membrane perturbing agent, EDTA and the antibiotics doxycycline, tigecycline, colistin, and chloramphenicol, respectively. Treatment of bacteria with [Formula: see text] plus EDTA, however, had the most dramatic and synergistic effect, with virtually all bacteria killed by 10 mM [Formula: see text], and EDTA (1 mM, aerobic, anaerobic). Most importantly, the PA4455 mutant was also sensitive to [Formula: see text] in biofilms. [Formula: see text] sensitivity and an anaerobic growth defect was also noted in two mutants (rmlC and wbpM) that are defective in B-band LPS synthesis, potentially indicating a membrane defect in the PA4455 mutant. Finally, this study describes a gene, PA4455, that when mutated, allows for dramatic sensitivity to the potential therapeutic agent, [Formula: see text] as well as EDTA. Furthermore, the synergy between the two compounds could offer future benefits against antibiotic resistant PA strains.
RESUMEN
The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that P. aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing ΔbdlA biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of P. aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing ΔbdlA and ΔdipA biofilms compared to wild-type planktonic cells. Despite this, bdlA and dipA inactivation, resulting in an inability to disperse in vitro, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, bdlA inactivation rendered P. aeruginosa more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Hidrolasas Diéster Fosfóricas/metabolismo , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Enfermedad Aguda , Animales , Proteínas Bacterianas/genética , Células Inmovilizadas/enzimología , Células Inmovilizadas/fisiología , Enfermedad Crónica , Eliminación de Gen , Interacciones Huésped-Patógeno , Pulmón/microbiología , Ratones , Interacciones Microbianas , Infecciones Oportunistas/microbiología , Hidrolasas Diéster Fosfóricas/genética , Plancton/crecimiento & desarrollo , Plancton/patogenicidad , Plancton/fisiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced â¼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd â¼6 µM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.
Asunto(s)
Catalasa/metabolismo , Óxido Nítrico/metabolismo , Pseudomonas aeruginosa/metabolismo , Anaerobiosis/efectos de los fármacos , Antibacterianos/farmacología , Catalasa/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Hierro/metabolismo , Nitritos/metabolismo , Óxidos de Nitrógeno/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Transcripción Genética/efectos de los fármacosRESUMEN
High-throughput screening (HTS) of 42 865 compounds was performed to identify compounds that inhibit formation of or kill Staphylococcus epidermidis RP62a biofilms. Three biological processes were assayed, including (1) growth of planktonic/biofilm bacteria, (2) assessment of metabolically active biofilm bacteria using a resazurin assay, and (3) assessment of biofilm biomass by crystal violet staining. After completing the three tiers (primary screening, hit confirmation, and dose-response curves), 352 compounds (representing ~0.8%) were selected as confirmed hit compounds from the HTS assay. The compounds were divided into groups based on their effectiveness on S. epidermidis biofilm properties. The majority of these affected both inhibition and killing of bacterial biofilm cultures. Only 16 of the confirmed hit compounds that have either an AC50 lower than 10 µM and/or Sconst ≥70 from those processed were selected for further study by confocal laser scanning microscopy (CLSM). The CLSM was used to evaluate the confirmed hit compounds on (1) inhibition of biofilm formation and (2) killing of preexisting S. epidermidis biofilms. Taken together, with further testing (e.g., disease-related conditions), such compounds may have applications as broad antimicrobial/antibiofilm use for prophylactic or therapeutic intervention to combat infections in surgical and intensive care clinics and battlefield settings.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Sensibilidad Microbiana/métodos , Staphylococcus epidermidis/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Microscopía Confocal , Bibliotecas de Moléculas Pequeñas , Staphylococcus epidermidis/fisiologíaRESUMEN
The reactivity of capsular extracellular polymeric substances (EPS) to chlorine and monochloramine was assessed and compared in this study. The impact of capsular EPS on Gram-negative bacteria Pseudomonas aeruginosa inactivation mechanisms was investigated both qualitatively and quantitatively using a combination of batch experiments, viability tests with LIVE/DEAD staining, and Fourier transform infrared spectroscopy (FTIR). Both wild-type and isogenic mutant strains with different alginate EPS production capabilities were used to evaluate their susceptibility to chlorine and monochloramine. The mucA22 mutant strain, which overproduces the EPS composed largely of acidic polysaccharide alginate, exhibited high resistance and prolonged inactivation time to both chlorine and monochloramine relative to PAO1 (wild-type) and algT(U) mutant strains (alginate EPS deficient). Multiple analyses were combined to better understand the mechanistic role of EPS against chlorine-based disinfectants. The extracted EPS exhibited high reactivity with chlorine and very low reactivity with monochloramine, suggesting different mechanism of protection against disinfectants. Moreover, capsular EPS on cell membrane appeared to reduce membrane permeabilization by disinfectants as suggested by deformation of key functional groups in EPS and cell membrane (the C-O-C stretching of carbohydrate and the C=O stretching of ester group). The combined results supported that capsular EPS, acting either as a disinfectant consumer (for chlorine inactivation) or limiting access to reactive sites on cell membrane (for monochloramine inactivation), provide a protective role for bacterial cells against regulatory residual disinfectants by reducing membrane permeabilization.
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Alginatos/química , Cápsulas Bacterianas/química , Cloraminas/farmacología , Cloro/farmacología , Desinfectantes/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Permeabilidad de la Membrana Celular , Espacio Extracelular/química , Viabilidad Microbiana , Modelos Estadísticos , Polímeros/química , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/fisiología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Most bacteria control oxidative stress through the H(2)O(2)-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that â¼70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo/genética , Pseudomonas aeruginosa/genética , Transactivadores/metabolismo , Sitios de Unión , Inmunoprecipitación de Cromatina , Genoma Bacteriano , Análisis de Secuencia por Matrices de Oligonucleótidos , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/metabolismo , Regulón , Tiorredoxinas/metabolismoRESUMEN
A hallmark of airways in patients with cystic fibrosis (CF) is highly refractory, chronic infections by several opportunistic bacterial pathogens. A recent study demonstrated that acidified sodium nitrite (A-NO(2)(-)) killed the highly refractory mucoid form of Pseudomonas aeruginosa, a pathogen that significantly compromises lung function in CF patients (S. S. Yoon et al., J. Clin. Invest. 116:436-446, 2006). Therefore, the microbicidal activity of A-NO(2)(-) (pH 6.5) against the following three major CF pathogens was assessed: P. aeruginosa (a mucoid, mucA22 mutant and a sequenced nonmucoid strain, PAO1), Staphylococcus aureus USA300 (methicillin resistant), and Burkholderia cepacia, a notoriously antibiotic-resistant organism. Under planktonic, anaerobic conditions, growth of all strains except for P. aeruginosa PAO1 was inhibited by 7.24 mM (512 µg ml(-1) NO(2)(-)). B. cepacia was particularly sensitive to low concentrations of A-NO(2)(-) (1.81 mM) under planktonic conditions. In antibiotic-resistant communities known as biofilms, which are reminiscent of end-stage CF airway disease, A-NO(2)(-) killed mucoid P. aeruginosa, S. aureus, and B. cepacia; 1 to 2 logs of cells were killed after a 2-day incubation with a single dose of â¼15 mM A-NO(2)(-). Animal toxicology and phase I human trials indicate that these bactericidal levels of A-NO(2)(-) can be easily attained by aerosolization. Thus, in summary, we demonstrate that A-NO(2)(-) is very effective at killing these important CF pathogens and could be effective in other infectious settings, particularly under anaerobic conditions where bacterial defenses against the reduction product of A-NO(2)(-), nitric oxide (NO), are dramatically reduced.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Burkholderia cepacia/efectos de los fármacos , Fibrosis Quística/microbiología , Plancton/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Nitrito de Sodio/farmacología , Anaerobiosis , Pruebas de Sensibilidad Microbiana , Microscopía ConfocalRESUMEN
The role of the Pseudomonas aeruginosa OxyR-controlled antioxidants alkyl hydroperoxide reductase CF (AhpCF) and catalase B (KatB) was evaluated in biofilm vs. planktonic culture upon exposure to hydrogen peroxide. AhpCF was found to be critical for survival of biofilm bacteria while KatB was more important for survival of planktonic free-swimming organisms.
Asunto(s)
Biopelículas , Catalasa/metabolismo , Peróxido de Hidrógeno/farmacología , Peroxirredoxinas/metabolismo , Plancton , Pseudomonas aeruginosa/efectos de los fármacos , Transactivadores/metabolismo , Antioxidantes/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Catalasa/efectos de los fármacos , Catalasa/genética , Medios de Cultivo , Regulación Bacteriana de la Expresión Génica , Peroxirredoxinas/efectos de los fármacos , Peroxirredoxinas/genética , Plancton/efectos de los fármacos , Plancton/enzimología , Plancton/crecimiento & desarrollo , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiologíaRESUMEN
OxyR controls H(2)O(2)-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<10(7)/ml) organisms are easily killed by micromolar H(2)O(2). The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H(2)O(2). We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H(2)O(2) than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H(2)O(2). Given the sensitivity of oxyR oprL bacteria to planktonic H(2)O(2), we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H(2)O(2)-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H(2)O(2). Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H(2)O(2) in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.
Asunto(s)
Biopelículas/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Peróxido de Hidrógeno/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Regulación Bacteriana de la Expresión Génica , Lipoproteínas/metabolismo , Microscopía Electrónica de Transmisión , Peptidoglicano/metabolismo , Plancton/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transactivadores/genéticaRESUMEN
The Xanthomonas campestris transcription regulator OhrR contains a reactive cysteine residue (C22) that upon oxidation by organic hydroperoxides (OHPs) forms an intersubunit disulphide bond with residue C127'. Such modification induces the expression of a peroxidase that reduces OHPs to their less toxic alcohols. Here, we describe the structures of reduced and OHP-oxidized OhrR, visualizing the structural mechanism of OHP induction. Reduced OhrR takes a canonical MarR family fold with C22 and C127' separated by 15.5 A. OHP oxidation results in the disruption of the Y36'-C22-Y47' interaction network and dissection of helix alpha5, which then allows the 135 degrees rotation and 8.2 A translation of C127', formation of the C22-C127' disulphide bond, and alpha6-alpha6' helix-swapped reconfiguration of the dimer interface. These changes result in the 28 degrees rigid body rotations of each winged helix-turn-helix motif and DNA dissociation. Similar effector-induced rigid body rotations are expected for most MarR family members.