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1.
J Bacteriol ; 204(7): e0061721, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35735992

RESUMO

Staphylococcus aureus is a medically important pathogen with high metabolic versatility allowing it to infect various niches within a host. S. aureus utilizes two major transcriptional regulators, namely, CodY and CcpA, to remodel metabolic and virulence gene expression in response to changing environmental conditions. Previous studies revealed that inactivation of either codY or ccpA has a pronounced impact on different aspects of staphylococcal physiology and pathogenesis. To determine the contribution and interplay of these two regulators in modulating central metabolism, virulence, and biofilm development, we constructed and characterized the codY ccpA double mutant in S. aureus UAMS-1. In line with previous studies, we found that CcpA and CodY control the cellular metabolic status by altering carbon flux through the central and overflow metabolic pathways. Our results demonstrate that ccpA inactivation impairs biofilm formation and decreases incorporation of extracellular DNA (eDNA) into the biofilm matrix, whereas disrupting codY resulted in a robust structured biofilm tethered together with eDNA and polysaccharide intercellular adhesin (PIA). Interestingly, inactivation of both codY and ccpA decreases biofilm biomass and reduces eDNA release in the double mutant. Compared with the inactivation of codY, the codY ccpA mutant did not overexpress toxins but maintained overexpression of amino acid metabolism pathways. Furthermore, the codY ccpA mutant produced large amounts of PIA, in contrast to the wild-type strain and ccpA mutant. Combined, the results of this study suggest that the coordinated action of CodY and CcpA modulate central metabolism, virulence gene expression, and biofilm-associated genes to optimize growth on preferred carbon sources until starvation sets in. IMPORTANCE Staphylococcus aureus is a leading cause of biofilm-associated infections, including infective endocarditis, worldwide. A greater understanding of metabolic forces driving biofilm formation in S. aureus is essential for the identification of novel therapeutic targets and for the development of new strategies to combat this medically important pathogen. This study characterizes the interplay and regulation of central metabolism and biofilm development by two global transcriptional regulators, CodY and CcpA. We found that the lack of CcpA and/or CodY have different impacts on intracellular metabolic status leading to a formation of morphologically altered biofilms. Overall, the results of this study provide new insights into our understanding of metabolism-mediated regulation of biofilm development in S. aureus.


Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Regulação Bacteriana da Expressão Gênica , Humanos , Staphylococcus aureus/metabolismo
2.
Anal Chem ; 94(5): 2615-2624, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35073053

RESUMO

Bacterial infections are the leading cause of morbidity and mortality in the world, particularly due to a delay in treatment and misidentification of the bacterial species causing the infection. Therefore, rapid and accurate identification of these pathogens has been of prime importance. The conventional diagnostic techniques include microbiological, biochemical, and genetic analyses, which are time-consuming, require large sample volumes, expensive equipment, reagents, and trained personnel. In response, we have now developed a paper-based ratiometric fluorescent sensor array. Environment-sensitive fluorescent dyes (3-hydroxyflavone derivatives) pre-adsorbed on paper microzone plates fabricated using photolithography, upon interaction with bacterial cell envelopes, generate unique fluorescence response patterns. The stability and reproducibility of the sensor array response were thoroughly investigated, and the analysis procedure was refined for optimal performance. Using neural networks for response pattern analysis, the sensor was able to identify 16 bacterial species and recognize their Gram status with an accuracy rate greater than 90%. The paper-based sensor was stable for up to 6 months after fabrication and required 30 times lower dye and sample volumes as compared to the analogous solution-based sensor. Therefore, this approach opens avenues to a state-of-the-art diagnostic tool that can be potentially translated into clinical applications in low-resource environments.


Assuntos
Bactérias , Infecções Bacterianas , Corantes Fluorescentes , Humanos , Reprodutibilidade dos Testes , Espectrometria de Fluorescência
3.
J Bacteriol ; 203(9)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33593944

RESUMO

Under conditions of glucose excess, aerobically growing bacteria predominantly direct carbon flux towards acetate fermentation, a phenomenon known as overflow metabolism or the bacterial 'Crabtree effect'. Numerous studies of the major acetate-generating pathway, the Pta-AckA, revealed its important role in bacterial fitness through the control of central metabolism to sustain balanced growth and cellular homeostasis. In this work, we highlight the contribution of the Pta-AckA pathway to fitness of the spore-forming bacterium, Bacillus anthracis We demonstrate that disruption of the Pta-AckA pathway causes a drastic growth reduction in the mutants and alters the metabolic and energy status of the cells. Our results revealed that inactivation of the Pta-AckA pathway increases the glucose consumption rate, affects intracellular ATP, NAD+ and NADH levels and leads to a metabolic block at the pyruvate and acetyl-CoA nodes. Consequently, accumulation of intracellular acetyl-CoA and pyruvate forces bacteria to direct carbon into the TCA and/or glyoxylate cycles as well as fatty acid and poly(3-hydroxybutyrate) (PHB) biosynthesis pathways. Notably, the presence of phosphate butyryltransferase in B. anthracis partially compensates for the loss of phosphotransacetylase activity. Furthermore, overexpression of the ptb gene not only eliminates the negative impact of the pta mutation on B. anthracis fitness, but also restores normal growth in the pta mutant of the non-butyrate-producing bacterium, Staphylococcus aureus Taken together, the results of this study demonstrate the importance of the Pta-AckA pathway for B. anthracis fitness by revealing its critical contribution to the maintenance of metabolic homeostasis during aerobic growth under conditions of carbon overflow.IMPORTANCE B. anthracis, the etiologic agent of anthrax, is a highly pathogenic, spore-forming bacterium that causes acute, life-threatening disease in both humans and livestock. A greater understanding of the metabolic determinants governing fitness of B. anthracis is essential for the development of successful therapeutic and vaccination strategies aimed at lessening the potential impact of this important biodefense pathogen. This study is the first to demonstrate the vital role of the Pta-AckA pathway in preserving energy and metabolic homeostasis in B. anthracis under conditions of carbon overflow, therefore, highlighting this pathway as a potential therapeutic target for drug discovery. Overall, the results of this study provide important insight into understanding the metabolic processes and requirements driving rapid B. anthracis proliferation during vegetative growth.

4.
J Bacteriol ; 202(8)2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32015143

RESUMO

The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm.IMPORTANCEStaphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , DNA Bacteriano/metabolismo , Matriz Extracelular/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/fisiologia , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Matriz Extracelular/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Polissacarídeos Bacterianos/genética , Proteínas Repressoras/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
5.
J Bacteriol ; 201(23)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31501288

RESUMO

The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of P cidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression.IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Staphylococcus aureus/metabolismo , Transcrição Gênica
6.
J Bacteriol ; 200(8)2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29378891

RESUMO

Staphylococcus aureus subverts innate defenses during infection in part by killing host immune cells to exacerbate disease. This human pathogen intercepts host cues and activates a transcriptional response via the S. aureus exoprotein expression (SaeR/SaeS [SaeR/S]) two-component system to secrete virulence factors critical for pathogenesis. We recently showed that the transcriptional repressor CodY adjusts nuclease (nuc) gene expression via SaeR/S, but the mechanism remained unknown. Here, we identified two CodY binding motifs upstream of the sae P1 promoter, which suggested direct regulation by this global regulator. We show that CodY shares a binding site with the positive activator SaeR and that alleviating direct CodY repression at this site is sufficient to abrogate stochastic expression, suggesting that CodY represses sae expression by blocking SaeR binding. Epistasis experiments support a model that CodY also controls sae indirectly through Agr and Rot-mediated repression of the sae P1 promoter. We also demonstrate that CodY repression of sae restrains production of secreted cytotoxins that kill human neutrophils. We conclude that CodY plays a previously unrecognized role in controlling virulence gene expression via SaeR/S and suggest a mechanism by which CodY acts as a master regulator of pathogenesis by tying nutrient availability to virulence gene expression.IMPORTANCE Bacterial mechanisms that mediate the switch from a commensal to pathogenic lifestyle are among the biggest unanswered questions in infectious disease research. Since the expression of most virulence genes is often correlated with nutrient depletion, this implies that virulence is a response to the lack of nourishment in host tissues and that pathogens like S. aureus produce virulence factors in order to gain access to nutrients in the host. Here, we show that specific nutrient depletion signals appear to be funneled to the SaeR/S system through the global regulator CodY. Our findings reveal a strategy by which S. aureus delays the production of immune evasion and immune-cell-killing proteins until key nutrients are depleted.


Assuntos
Proteínas de Bactérias/metabolismo , Nutrientes , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Células Cultivadas , Meios de Cultura/química , Regulação Bacteriana da Expressão Gênica , Humanos , Leucocidinas/metabolismo , Neutrófilos/microbiologia , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Staphylococcus aureus/genética , Fatores de Transcrição/genética , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
7.
J Bacteriol ; 200(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712876

RESUMO

In Staphylococcus aureus, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY-regulated targets. In this work, we investigated the impact of guanine nucleotides (GNs) on S. aureus physiology and CodY activity by constructing a guaA null mutant (ΔguaA strain). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the ΔguaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaA cells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of the S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.IMPORTANCEStaphylococcus aureus infections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of S. aureus in response to environmental cues is critical for developing efficient diagnostics and treatments. De novo purine biosynthesis is essential for both fitness and virulence in S. aureus since inhibiting production cripples S. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects S. aureus fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat S. aureus infections.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Guanina/administração & dosagem , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Biofilmes , Genótipo , Guanina/metabolismo , Guanina/farmacologia , Guanosina/administração & dosagem , Guanosina/metabolismo , RNA Bacteriano , Proteínas Repressoras/genética , Análise de Sequência de RNA , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Transcriptoma , Fatores de Virulência
8.
Mol Microbiol ; 104(5): 793-803, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28299860

RESUMO

Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species.


Assuntos
Bacillus anthracis/metabolismo , Hidroxibutiratos/metabolismo , Lipídeos/biossíntese , Poliésteres/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Bacillus anthracis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Ácidos Picolínicos/metabolismo , Esporos Bacterianos/metabolismo
9.
Mol Microbiol ; 101(3): 495-514, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27116338

RESUMO

The global regulator CodY controls the expression of dozens of metabolism and virulence genes in the opportunistic pathogen Staphylococcus aureus in response to the availability of isoleucine, leucine and valine (ILV), and GTP. Using RNA-Seq transcriptional profiling and partial activity variants, we reveal that S. aureus CodY activity grades metabolic and virulence gene expression as a function of ILV availability, mediating metabolic reorganization and controlling virulence factor production in vitro. Strains lacking CodY regulatory activity produce a PIA-dependent biofilm, but development is restricted under conditions that confer partial CodY activity. CodY regulates the expression of thermonuclease (nuc) via the Sae two-component system, revealing cascading virulence regulation and factor production as CodY activity is reduced. Proteins that mediate the host-pathogen interaction and subvert the immune response are shut off at intermediate levels of CodY activity, while genes coding for enzymes and proteins that extract nutrients from tissue, that kill host cells, and that synthesize amino acids are among the last genes to be derepressed. We conclude that S. aureus uses CodY to limit host damage to only the most severe starvation conditions, providing insight into one potential mechanism by which S. aureus transitions from a commensal bacterium to an invasive pathogen.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Biofilmes , Interações Hospedeiro-Patógeno/genética , Staphylococcus aureus/metabolismo , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
10.
Mol Microbiol ; 101(6): 942-53, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27253847

RESUMO

The Staphylococcus aureus LysR-type transcriptional regulator, CidR, activates the expression of two operons including cidABC and alsSD that display pro- and anti-death functions, respectively. Although several investigations have focused on the functions of different genes associated with these operons, the collective role of the CidR regulon in staphylococcal physiology is not clearly understood. Here we reveal that the primary role of this regulon is to limit acetate-dependent potentiation of cell death in staphylococcal populations. Although both CidB and CidC promote acetate generation and cell death, the CidR-dependent co-activation of CidA and AlsSD counters the effects of CidBC by redirecting intracellular carbon flux towards acetoin formation. From a mechanistic standpoint, we demonstrate that CidB is necessary for full activation of CidC, whereas CidA limits the abundance of CidC in the cell.


Assuntos
Proteínas de Bactérias/genética , Staphylococcus aureus/citologia , Staphylococcus aureus/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Óperon , Elementos Reguladores de Transcrição , Regulon , Staphylococcus aureus/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica
11.
J Proteome Res ; 15(4): 1205-12, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26975873

RESUMO

The phosphotransacetylase-acetate kinase (Pta-AckA) pathway is thought to be a vital ATP generating pathway for Staphylococcus aureus. Disruption of the Pta-AckA pathway during overflow metabolism causes significant reduction in growth rate and viability, albeit not due to intracellular ATP depletion. Here, we demonstrate that toxicity associated with inactivation of the Pta-AckA pathway resulted from an altered intracellular redox environment. Growth of the pta and ackA mutants under anaerobic conditions partially restored cell viability. NMR metabolomics analyses and (13)C6-glucose metabolism tracing experiments revealed the activity of multiple pathways that promote redox (NADH/NAD(+)) turnover to be enhanced in the pta and ackA mutants during anaerobic growth. Restoration of redox homeostasis in the pta mutant by overexpressing l- lactate dehydrogenase partially restored its viability under aerobic conditions. Together, our findings suggest that during overflow metabolism, the Pta-AckA pathway plays a critical role in preventing cell viability defects by promoting intracellular redox homeostasis.


Assuntos
Acetato Quinase/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Metabolômica , Fosfato Acetiltransferase/genética , Staphylococcus aureus/genética , Acetato Quinase/deficiência , Trifosfato de Adenosina/biossíntese , Aerobiose , Anaerobiose , Proteínas de Bactérias/metabolismo , Isótopos de Carbono , Glucose/metabolismo , Homeostase , L-Lactato Desidrogenase/metabolismo , Espectroscopia de Ressonância Magnética , Viabilidade Microbiana , Mutação , NAD/metabolismo , Oxirredução , Fosfato Acetiltransferase/deficiência , Staphylococcus aureus/metabolismo
12.
Mol Microbiol ; 95(4): 723-37, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25491472

RESUMO

Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically localized within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, overactivation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events.


Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Transdução de Sinais , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Fatores de Transcrição/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Óperon , Mutação Puntual , Regiões Promotoras Genéticas , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica
13.
PLoS Pathog ; 10(6): e1004205, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24945831

RESUMO

Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC) and α-acetolactate synthase/decarboxylase (AlsSD) overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.


Assuntos
Acetolactato Sintase/metabolismo , Biofilmes/crescimento & desenvolvimento , Carboxiliases/metabolismo , Piruvato Oxidase/metabolismo , Staphylococcus aureus/metabolismo , Acetatos/metabolismo , Animais , Carbono/metabolismo , Dano ao DNA , Endocardite Bacteriana/imunologia , Endocardite Bacteriana/microbiologia , Endocardite Bacteriana/patologia , Regulação Bacteriana da Expressão Gênica , Consumo de Oxigênio , Coelhos , Espécies Reativas de Oxigênio
14.
PLoS Pathog ; 8(11): e1003033, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209408

RESUMO

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced (13)C(5) labeled arginine when grown with (13)C(5) proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo.


Assuntos
Abscesso/metabolismo , Proteínas de Bactérias/metabolismo , Nefropatias/metabolismo , Prolina/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Transcrição/metabolismo , Abscesso/genética , Abscesso/microbiologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Genoma Bacteriano/genética , Nefropatias/genética , Nefropatias/microbiologia , Camundongos , Mutação , Prolina/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Fatores de Transcrição/genética
15.
J Bacteriol ; 195(13): 3035-44, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23625849

RESUMO

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD(+), and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism.


Assuntos
Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico/genética , Ciclo do Ácido Cítrico/fisiologia , Glicólise/genética , Glicólise/fisiologia , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Staphylococcus aureus/genética
16.
Appl Environ Microbiol ; 79(11): 3413-24, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23524683

RESUMO

The Staphylococcus aureus cid and lrg operons play significant roles in the control of autolysis and accumulation of extracellular genomic DNA (eDNA) during biofilm development. Although the molecular mechanisms mediating this control are only beginning to be revealed, it is clear that cell death must be limited to a subfraction of the biofilm population. In the present study, we tested the hypothesis that cid and lrg expression varies during biofilm development as a function of changes in the availability of oxygen. To examine cid and lrg promoter activity during biofilm development, fluorescent reporter fusion strains were constructed and grown in a BioFlux microfluidic system, generating time-lapse epifluorescence images of biofilm formation, which allows the spatial and temporal localization of gene expression. Consistent with cid induction under hypoxic conditions, the cid::gfp fusion strain expressed green fluorescent protein predominantly within the interior of the tower structures, similar to the pattern of expression observed with a strain carrying a gfp fusion to the hypoxia-induced promoter controlling the expression of the lactose dehydrogenase gene. The lrg promoter was also expressed within towers but appeared more diffuse throughout the tower structures, indicating that it was oxygen independent. Unexpectedly, the results also demonstrated the existence of tower structures with different expression phenotypes and physical characteristics, suggesting that these towers exhibit different metabolic activities. Overall, the findings presented here support a model in which oxygen is important in the spatial and temporal control of cid expression within a biofilm and that tower structures formed during biofilm development exhibit metabolically distinct niches.


Assuntos
Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/genética , Técnicas Analíticas Microfluídicas/métodos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Oxigênio/metabolismo , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase em Tempo Real , Staphylococcus aureus/genética
17.
J Bacteriol ; 193(22): 6187-96, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21926234

RESUMO

Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant synthesis, the rpiR homologues were inactivated, and the effects on PPP activity and virulence factor synthesis were assessed. Two of the three homologues (RpiRB and RpiRC) positively influence the transcription of the PPP genes rpiA and zwf, while the third homologue (RpiRA) is slightly antagonistic to the other homologues. In addition, inactivation of RpiRC altered the temporal transcription of RNAIII, the effector molecule of the agr quorum-sensing system. These data confirm the close linkage of central metabolism and virulence determinant synthesis, and they establish a metabolic override for quorum-sensing-dependent regulation of RNAIII transcription.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Via de Pentose Fosfato , RNA Bacteriano/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , RNA Bacteriano/genética , Staphylococcus aureus/genética , Fatores de Transcrição/genética
18.
J Proteome Res ; 10(8): 3743-54, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21692534

RESUMO

We previously hypothesized that Staphylococcus epidermidis senses a diverse set of environmental and nutritional factors associated with biofilm formation through a modulation in the activity of the tricarboxylic acid (TCA) cycle. Herein, we report our further investigation of the impact of additional environmental stress factors on TCA cycle activity and provide a detailed description of our NMR methodology. S. epidermidis wild-type strain 1457 was treated with stressors that are associated with biofilm formation, a sublethal dose of tetracycline, 5% NaCl, 2% glucose, and autoinducer-2 (AI-2). As controls and to integrate our current data with our previous study, 4% ethanol stress and iron-limitation were also used. Consistent with our prior observations, the effect of many environmental stress factors on the S. epidermidis metabolome was essentially identical to the effect of TCA cycle inactivation in the aconitase mutant strain 1457-acnA::tetM. A detailed quantitative analysis of metabolite concentration changes using 2D (1)H-(13)C HSQC and (1)H-(1)H TOCSY spectra identified a network of 37 metabolites uniformly affected by the stressors and TCA cycle inactivation. We postulate that the TCA cycle acts as the central pathway in a metabolic signaling network.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Transdução de Sinais , Staphylococcus epidermidis/metabolismo , Biofilmes , Ciclo do Ácido Cítrico , Metaboloma , Análise de Componente Principal , Staphylococcus epidermidis/crescimento & desenvolvimento
19.
J Biol Chem ; 285(47): 36616-24, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20861019

RESUMO

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to "sense" environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is "sensing" disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are "sensed" by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Espectroscopia de Ressonância Magnética , Metabolômica , Staphylococcus epidermidis/metabolismo , Proteínas de Bactérias/genética , Northern Blotting , Depressores do Sistema Nervoso Central/farmacologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Etanol/farmacologia , Deficiências de Ferro , RNA Bacteriano/genética , Transdução de Sinais
20.
Microbiology (Reading) ; 157(Pt 12): 3458-3468, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21964732

RESUMO

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR-LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Ciclo do Ácido Cítrico/genética , Metabolismo Energético , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Staphylococcus epidermidis/fisiologia , Animais , Proteínas de Bactérias/genética , Feminino , Deleção de Genes , Humanos , Camundongos , Polissacarídeos Bacterianos/metabolismo , Proteínas Repressoras/genética , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/metabolismo , Virulência
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