RESUMO
The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted catabolite-responsive element (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection.IMPORTANCE Hospital-acquired infections account for 2 billion dollars annually in increased health care expenses and cause more than 100,000 deaths in the United States alone. Enterococci are the second leading cause of hospital-acquired infections. They form biofilms at surgical sites and are often associated with infections of the urinary tract following catheterization. Nutrient uptake and growth are key factors that influence their ability to cause disease. Our research identified a large set of genes that illuminate nutrient uptake pathways in enterococci. Perturbation of the metabolic circuit reduces virulence in a rabbit endocarditis model, as well as in catheter-associated urinary tract infection in mice. Targeting metabolic pathways that are important in infection may lead to new treatments against multidrug-resistant enterococcal infections.
Assuntos
Proteínas de Bactérias/genética , Carbono/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Expressão Gênica , Fator sigma/genética , Animais , Proteínas de Bactérias/metabolismo , Biofilmes , Regulação Bacteriana da Expressão Gênica , Coelhos , Fator sigma/classificação , VirulênciaRESUMO
Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen Aggregatibacter actinomycetemcomitans One of this bacterium's key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about A. actinomycetemcomitans since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on A. actinomycetemcomitans is hampered by the field's lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of A. actinomycetemcomitans, revealing a core set of 319 genes. We then generated an arrayed mutant library comprising >1,500 unique insertions and used a sequencing-based approach to define each mutant's position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces A. actinomycetemcomitans to form biofilms. We therefore devised a novel Tn-seq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen.IMPORTANCE Millions suffer from gum disease, which often is caused by Aggregatibacter actinomycetemcomitans, a bacterium that forms antibiotic-resistant biofilms. To fully understand any organism, we should be able to answer: what genes does it require for life? Here, we address this question for A. actinomycetemcomitans by determining the genes in its genome that cannot be mutated. As for the genes that can be mutated, we archived these mutants into a library, which we used to find genes that contribute to antibiotic resistance, leading us to discover that antibiotics cause A. actinomycetemcomitans to form biofilms. We then devised an approach to find genes that mediate this process and confirmed 4 genes. These results illuminate new fundamental traits of a human pathogen.
Assuntos
Aggregatibacter actinomycetemcomitans/genética , Genoma Bacteriano , Periodontite/microbiologia , Aggregatibacter actinomycetemcomitans/classificação , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Biblioteca Gênica , Aptidão Genética , Genômica , Humanos , Boca/microbiologia , Mutagênese InsercionalRESUMO
Staphylococcus aureus-human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species.
RESUMO
Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains.
Assuntos
Enterococcus faecalis/efeitos dos fármacos , Feromônios/farmacologia , Farmacorresistência Bacteriana Múltipla , HumanosRESUMO
Microarrays were used to investigate the transcriptional response of Enterococcus faecalis to photostress. E. faecalis are Gram-positive bacteria used as indicators of water quality and have been shown to vary diurnally in response to sunlight. E. faecalis in filtered seawater microcosms were exposed to artificial sunlight for 12h and then placed in the dark for 12h. Transcript abundance was measured at 0, 2, 6, 12, and 24h in the sunlit microcosm and a dark control using microarrays. Culturable E. faecalis concentrations decreased 6-7 orders of magnitude within the first 6h of light exposure. After 12h in the dark, no evidence of dark-repair was observed. Expression data collected after 12h of sunlight exposure revealed a difference in transcript abundance in the light relative to dark microcosms for 35 unique ORFs, 33 ORFs showed increased transcript abundance and 2 ORFs showed reduced transcript abundance. A majority (51%) of the ORFs with increased transcript abundance in the sunlit relative to dark microcosms encoded hypothetical proteins; others were associated with protein synthesis, oxidative stress and DNA repair. Results suggest that E. faecalis exposed to sunlight actively transcribe RNA in response to photostress.
Assuntos
Enterococcus faecalis/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz Solar/efeitos adversos , Proteínas de Bactérias/genética , Dano ao DNA , Reparo do DNA , Enterococcus faecalis/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética , Transcrição Gênica/efeitos da radiaçãoRESUMO
Ultrafiltration, nanofiltration membranes and feed spacers were hydrophilized with polydopamine and polydopamine-g-poly(ethylene glycol) surface coatings. The fouling propensity of modified and unmodified membranes was evaluated by short-term batch protein and bacterial adhesion tests. The fouling propensity of modified and unmodified membranes and spacers was evaluated by continuous biofouling experiments in a membrane fouling simulator. The goals of the study were: 1) to determine the effectiveness of polydopamine and polydopamine-g-poly(ethylene glycol) membrane coatings for biofouling control and 2) to compare techniques commonly used in assessment of membrane biofouling propensity with biofouling experiments under practical conditions. Short-term adhesion tests were carried out under static, no-flow conditions for 1 h using bovine serum albumin, a common model globular protein, and Pseudomonas aeruginosa, a common model Gram-negative bacterium. Biofouling tests were performed in a membrane fouling simulator (MFS) for several days under flow conditions similar to those encountered in industrial modules with the autochthonous drinking water population and acetate dosage as organic substrate. Polydopamine- and polydopamine-g-poly(ethylene glycol)-modified membranes showed significantly reduced adhesion of bovine serum albumin and P. aeruginosa in the short-term adhesion tests, but no reduction of biofouling was observed during longer biofouling experiments with modified membranes and spacers. These results demonstrate that short-term batch adhesion experiments using model proteins or bacteria under static conditions are not indicative of biofouling, while continuous biofouling experiments showed that membrane surface modification by polydopamine and polydopamine-g-poly(ethylene glycol) is not effective for biofouling control.
Assuntos
Incrustação Biológica , Indóis/análise , Membranas Artificiais , Polietilenoglicóis/análise , Polímeros/análise , Animais , Aderência Bacteriana , Bovinos , Indóis/química , Polietilenoglicóis/química , Polímeros/química , Pseudomonas aeruginosa/fisiologia , Albumina Sérica/química , Propriedades de SuperfícieRESUMO
Bacteria are social organisms that possess multiple pathways for sensing and responding to small molecules produced by other microbes. Most bacteria in nature exist in sessile communities called biofilms, and the ability of biofilm bacteria to sense and respond to small molecule signals and cues produced by neighboring biofilm bacteria is particularly important. To understand microbial interactions between biofilms, it is necessary to perform rapid, real-time spatial quantification of small molecules in microenvironments immediately surrounding biofilms; however, such measurements have been elusive. In this study, scanning electrochemical microscopy was used to quantify small molecules surrounding a biofilm in 3D space. Measuring concentrations of the redox-active signaling molecule pyocyanin (PYO) produced by biofilms of the bacterium Pseudomonas aeruginosa revealed a high concentration of PYO that is actively maintained in the reduced state proximal to the biofilm. This gradient results in a reduced layer of PYO that we have termed the PYO "electrocline," a gradient of redox potential, which extends several hundred microns from the biofilm surface. We also demonstrate that the PYO electrocline is formed under electron acceptor-limiting conditions, and that growth conditions favoring formation of the PYO electrocline correlate to an increase in soluble iron. Additionally, we have taken a "reactive image" of a biofilm surface, demonstrating the rate of bacterial redox activity across a 2D surface. These studies establish methodology for spatially coordinated concentration and redox status measurements of microbe-produced small molecules and provide exciting insights into the roles these molecules play in microbial competition and nutrient acquisition.
Assuntos
Biofilmes , Eletroquímica/métodos , Metaboloma , Pseudomonas aeruginosa/metabolismo , Ferro/metabolismo , Modelos Biológicos , Oxirredução , Piocianina/análise , Piocianina/química , Fatores de TempoRESUMO
Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.
Assuntos
Coinfecção/microbiologia , Ácido Láctico/metabolismo , Pasteurellaceae/patogenicidade , Streptococcus gordonii/patogenicidade , Abscesso/microbiologia , Animais , Técnicas de Cocultura , Glucose/metabolismo , Camundongos , Interações Microbianas , Mutação , Pasteurellaceae/genética , Pasteurellaceae/crescimento & desenvolvimento , Streptococcus gordonii/genética , Streptococcus gordonii/crescimento & desenvolvimentoRESUMO
Quantitative detection of hydrogen peroxide in solution above a Streptococcus gordonii (Sg) bacterial biofilm was studied in real time by scanning electrochemical microscopy (SECM). The concentration of hydrogen peroxide was determined to be 0.7 mM to 1.6 mM in the presence of 10 mM glucose over a period of 2 to 8 h. The hydrogen peroxide production measured was higher near the biofilm surface in comparison to Sg grown planktonically. Differential hydrogen peroxide production was observed both by fluorometric as well as by SECM measurements. The interaction between two different species in a bacterial biofilm of Sg and Aggregatibacter actinomycetemcomitans (Aa) in terms of hydrogen peroxide production was also studied by SECM. One-directional y-scan SECM measurements showed the unique spatial mapping of hydrogen peroxide concentration across a mixed species biofilm and revealed that hydrogen peroxide concentration varies greatly dependent upon local species composition.
Assuntos
Biofilmes , Eletroquímica/métodos , Peróxido de Hidrogênio/metabolismo , Microscopia Eletrônica de Varredura/métodos , Monitorização Fisiológica/métodos , Pasteurellaceae/metabolismo , Streptococcus gordonii/metabolismo , Simulação por Computador , Fluorometria , Peróxido de Hidrogênio/análise , Microeletrodos , Especificidade da EspécieRESUMO
Bacteria in the human oral cavity often grow in an attached multispecies biofilm community. Members of this community display defined interactions that have an impact on the physiology of the individual and the group. Here, we show that during coculture growth with streptococci, the oral pathogen Aggregatibacter actinomycetemcomitans displays enhanced resistance to killing by host innate immunity. The mechanism of resistance involves sensing of the streptococcal metabolite hydrogen peroxide by A. actinomycetemcomitans, which stimulates a genetic program resulting in enhanced expression of the complement resistance protein ApiA. The oxidative stress response regulator OxyR mediates induction of apiA transcription, and this induction is required for coculture resistance to killing by human serum. These findings provide evidence that interaction between community members mediates prokaryotic resistance to host innate immunity and reinforce the need to understand how polymicrobial growth affects interaction with the host immune system.
Assuntos
Imunidade Inata , Proteobactérias/fisiologia , Proteínas de Bactérias/fisiologia , Sequência de Bases , Sangue , Técnicas de Cocultura , Proteínas do Sistema Complemento/fisiologia , DNA Bacteriano , Humanos , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Estresse Oxidativo , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos dos fármacosRESUMO
Biofilm formation by Pseudomonas aeruginosa is hypothesized to follow a developmental pattern initiated by attachment to a surface followed by microcolony formation and mature biofilm development. Swimming and twitching motility are important for attachment and biofilm development in P. aeruginosa. However, it is clear that many P. aeruginosa strains lacking swimming motility exist as biofilms in the lungs of cystic fibrosis patients. Consequently, we have developed a dynamic attachment assay to identify motility-independent attachment-defective mutants. Using transposon mutagenesis, we identified 14 novel dynamic attachment-deficient (dad) mutants including four mutants specific to dynamic assay conditions (dad specific). Two of the dad-specific mutants contain insertions in genes involved in sensing and responding to external stimuli, implying a significant impact of external factors on the biofilm developmental pathway. Observations of initial attachment and long-term biofilm formation characterized our dad mutants into two distinct classes: biofilm delayed and biofilm impaired. Biofilm-delayed mutants form wild-type biofilms but are delayed at least 24 h compared with the wild type, whereas biofilm-impaired mutants never form wild-type biofilms in our assays. We propose a dynamic model for attachment and biofilm formation in P. aeruginosa including these two classes.
Assuntos
Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Mutação , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Fibrose Cística/microbiologia , Elementos de DNA Transponíveis , Humanos , Mutagênese Insercional/métodos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genéticaRESUMO
The response of Desulfovibrio vulgaris to Cu(II) and Hg(II) was characterized. Both metals increased the lag phase, and Cu(II) reduced cell yield at concentrations as low as 50 microM. mRNA expression was analyzed using random arbitrarily primed PCR, differential display, and quantitative PCR. Both Cu(II) and Hg(II) (50 micro M) caused upregulation of mRNA expression for an ATP binding protein (ORF2004) and an ATPase (ORF856) with four- to sixfold increases for Hg(II) and 1.4- to 3-fold increases with Cu(II). These results suggest that D. vulgaris uses an ATP-dependent mechanism for adapting to toxic metals in the environment.