RESUMO
In this work, we provide the first in vitro characterization of two essential proteins from Staphylococcus aureus (S. aureus) involved in iron-sulfur (Fe-S) cluster biogenesis: the cysteine desulfurase SufS and the sulfurtransferase SufU. Together, these proteins form the transient SufSU complex and execute the first stage of Fe-S cluster biogenesis in the SUF-like pathway in Gram-positive bacteria. The proteins involved in the SUF-like pathway, such as SufS and SufU, are essential in Gram-positive bacteria since these bacteria tend to lack redundant Fe-S cluster biogenesis pathways. Most previous work characterizing the SUF-like pathway has focused on Bacillus subtilis (B. subtilis). We focus on the SUF-like pathway in S. aureus because of its potential to serve as a therapeutic target to treat S. aureus infections. Herein, we characterize S. aureus SufS (SaSufS) by X-ray crystallography and UV-vis spectroscopy, and we characterize S. aureus SufU (SaSufU) by a zinc binding fluorescence assay and small-angle X-ray scattering. We show that SaSufS is a type II cysteine desulfurase and that SaSufU is a Zn2+-containing sulfurtransferase. Additionally, we evaluated the cysteine desulfurase activity of the SaSufSU complex and compared its activity to that of B. subtilis SufSU. Subsequent cross-species activity analysis reveals a surprising result: SaSufS is significantly less stimulated by SufU than BsSufS. Our results set a basis for further characterization of SaSufSU as well as the development of new therapeutic strategies for treating infections caused by S. aureus by inhibiting the SUF-like pathway.
RESUMO
Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas Ferro-Enxofre/biossíntese , Bactérias/genética , Proteínas de Bactérias/genética , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Enxofre/metabolismoRESUMO
Iron-sulfur (Fe-S) clusters are one of the most ubiquitous and versatile prosthetic groups exploited by nature. Fe-S clusters aid in conducting redox reactions, carbon activation, and environmental sensing. This chapter presents an overview of the genetic approaches that have been useful for identifying and characterizing bacterial factors involved in Fe-S protein assembly. Traditional genetic screens that assess viability or conditional auxotrophies and bioinformatic approaches have identified the majority of the described genes utilized for Fe-S protein assembly. Herein, we expand upon this list of genetic methods by detailing the use of transposon sequencing (TnSeq) to identify gene products that are necessary for the proper function of metabolic pathways that require Fe-S enzymes. TnSeq utilizes the power of genomics and massively parallel DNA sequencing to allow researchers to quantify the necessity of individual gene products for a specific growth condition. This allows for the identification of gene products or gene networks that have a role in a given metabolic process but are not essential for the process. An advantage of this approach is that it allows researchers to identify mutants that have partial phenotypes that are often missed using traditional plate-based selections. Applying TnSeq to address questions of Fe-S protein maturation will result in a more comprehensive understanding of genetic interactions and factors utilized in Fe-S biogenesis and Fe-S protein assembly.
Assuntos
Genômica , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Redes e Vias Metabólicas , Enxofre/metabolismoRESUMO
Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The ΔbshA Δsod and ΔbshA Δpcat double mutant strains were as sensitive to Hg(II) as the ΔbshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δsod and Δpcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δsod and Δpcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments.IMPORTANCEThermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus-Thermus phylum that, together with the Aquificae, constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243-249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321-331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes.
Assuntos
Catalase/metabolismo , Cisteína/análogos & derivados , Tolerância a Medicamentos , Glucosamina/análogos & derivados , Compostos de Mercúrio/toxicidade , Superóxido Dismutase/metabolismo , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/enzimologia , Catalase/genética , Cisteína/metabolismo , Deleção de Genes , Glucosamina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Recent advances in low-level light devices have opened new treatment options for mild to moderate acne patients. Light therapies have been used to treat a variety of skin conditions over the years but were typically only available as treatments provided by professional clinicians. Clinical application of blue light has proven to be effective for a broader spectral range and at lower fluences than previously utilized. Herein, we tested the hypothesis that sub-milliwatt/cm2 levels of long-wave blue light (449 nm) effectively kills Propionibacterium acnes, a causative agent of acne vulgaris, in vitro. MATERIALS AND METHODS: Two types of LED light boards were designed to facilitate in vitro blue light irradiation to either six-well plates containing fluid culture or a petri plate containing solid medium. P. acnes. Survival was determined by counting colony forming units (CFU) following irradiation. P. acnes was exposed in the presence and absence of oxygen. Coproporphyrin III (CPIII) photoexcitation was spectrophotometrically evaluated at 415 and 440 nm to compare the relative photochemical activities of these wavelengths. RESULTS: 422 and 449 nm blue light killed P. acnes in planktonic culture. Irradiation with 449 nm light also effectively killed P. acnes on a solid agar surface. Variation of time or intensity of light exposure resulted in a fluence-dependent improvement of antimicrobial activity. The presence of oxygen was necessary for killing of P. acnes with 449 nm light. CPIII displayed clear photoexcitation at both 415 and 440 nm, indicating that both wavelengths are capable of initiating CPIII photoexcitation at low incident light intensities (50 uW/cm2 ). CONCLUSION: Herein we demonstrate that sub-milliwatt/cm2 levels of long-wave blue light (449 nm) effectively kill P. acnes. The methods and results presented allow for deeper exploration and design of light therapy treatments. Results from these studies are expanding our understanding of the mode of action and functionality of blue light, allowing for improved options for acne patients. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.
Assuntos
Acne Vulgar/microbiologia , Acne Vulgar/radioterapia , Terapia com Luz de Baixa Intensidade/métodos , Propionibacterium acnes/efeitos da radiação , Humanos , Técnicas In Vitro , Estudos de Amostragem , Sensibilidade e EspecificidadeRESUMO
Comprehending biology at the molecular and systems levels is predicated upon understanding the functions of proteins. Proteins are typically composed of one or more functional moieties termed domains. Members of Bacteria, Eukarya, and Archaea utilize proteins containing a domain of unknown function (DUF) 59. Proteins requiring iron-sulfur (FeS) clusters containing cofactors are necessary for nearly all organisms making the assembly of functional FeS proteins essential. Recently, studies in eukaryotic and bacterial organisms have shown that proteins containing a DUF59, or those composed solely of DUF59, function in FeS protein maturation and/or intracellular Fe homeostasis. Herein, we review the current literature, discuss potential roles for DUF59, and address future studies that will help advance the field.
Assuntos
Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Eucariotos/metabolismo , Homeostase , Espaço Intracelular/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Relação Estrutura-AtividadeRESUMO
During times of environmental insult, Bacillus subtilis undergoes developmental changes leading to biofilm formation, sporulation and competence. Each of these states is regulated in part by the phosphorylated form of the master response regulator Spo0A (Spo0Aâ¼P). The phosphorylation state of Spo0A is controlled by a multi-component phosphorelay. RicA, RicF and RicT (previously YmcA, YlbF and YaaT) have been shown to be important regulatory proteins for multiple developmental fates. These proteins directly interact and form a stable complex, which has been proposed to accelerate the phosphorelay. Indeed, this complex is sufficient to stimulate the rate of phosphotransfer amongst the phosphorelay proteins in vitro. In this study, we demonstrate that two [4Fe-4S]2+ clusters can be assembled on the complex. As with other iron-sulfur cluster-binding proteins, the complex was also found to bind FAD, hinting that these cofactors may be involved in sensing the cellular redox state. This work provides the first comprehensive characterization of an iron-sulfur protein complex that regulates Spo0Aâ¼P levels. Phylogenetic and genetic evidence suggests that the complex plays a broader role beyond stimulation of the phosphorelay.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Cisteína/metabolismo , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/metabolismo , Oxirredução , Fosforilação , Filogenia , Esporos Bacterianos , Fatores de Transcrição/genéticaRESUMO
Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Neutrófilos/microbiologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Proteínas de Bactérias/genética , Humanos , Proteínas Ferro-Enxofre/genética , Oxigênio/metabolismo , RNA Antissenso/análise , Espécies Reativas de Nitrogênio/metabolismo , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , VirulênciaRESUMO
The rising problem of antimicrobial resistance in Staphylococcus aureus necessitates the discovery of novel therapeutic targets for small-molecule intervention. A major obstacle of drug discovery is identifying the target of molecules selected from high-throughput phenotypic assays. Here, we show that the toxicity of a small molecule termed '882 is dependent on the constitutive activity of the S. aureus virulence regulator SaeRS, uncovering a link between virulence factor production and energy generation. A series of genetic, physiological, and biochemical analyses reveal that '882 inhibits iron-sulfur (Fe-S) cluster assembly most likely through inhibition of the Suf complex, which synthesizes Fe-S clusters. In support of this, '882 supplementation results in decreased activity of the Fe-S cluster-dependent enzyme aconitase. Further information regarding the effects of '882 has deepened our understanding of virulence regulation and demonstrates the potential for small-molecule modulation of Fe-S cluster assembly in S. aureus and other pathogens.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Fatores de Virulência/metabolismo , Aconitato Hidratase/metabolismo , Antibacterianos/química , Descoberta de Drogas , Humanos , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Fatores de Transcrição/metabolismo , Virulência/efeitos dos fármacosRESUMO
Staphylococcus aureus SufT is composed solely of the domain of unknown function 59 (DUF59) and has a role in the maturation of iron-sulphur (Fe-S) proteins. We report that SufT is essential for S. aureus when growth is heavily reliant upon lipoamide-utilizing enzymes, but dispensable when this reliance is decreased. LipA requires Fe-S clusters for lipoic acid (LA) synthesis and a ΔsufT strain had phenotypes suggestive of decreased LA production and decreased activities of lipoamide-requiring enzymes. Fermentative growth, a null clpC allele, or decreased flux through the TCA cycle diminished the demand for LA and rendered SufT non-essential. Abundance of the Fe-S cluster carrier Nfu was increased in a ΔclpC strain and a null clpC allele was unable to suppress the LA requirement of a ΔsufT Δnfu strain. Over-expression of nfu suppressed the LA requirement of the ΔsufT strain. We propose a model wherein SufT, and by extension the DUF59, is essential for the maturation of holo-LipA in S. aureus cells experiencing a high demand for lipoamide-dependent enzymes. The findings presented suggest that the demand for products of Fe-S enzymes is a factor governing the usage of one Fe-S cluster assembly factor over another in the maturation of apo-proteins.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , Staphylococcus aureus/metabolismo , Aconitato Hidratase/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Proteínas de Escherichia coli , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Infecções Estafilocócicas , Staphylococcus aureus/genética , Enxofre/metabolismo , Ácido Tióctico/análogos & derivados , Ácido Tióctico/genética , Ácido Tióctico/metabolismo , Fatores de TranscriçãoRESUMO
Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins.
Assuntos
Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Proteínas Ferro-Enxofre/genética , Ferro/metabolismo , Infecções Estafilocócicas/genética , Staphylococcus aureus/metabolismo , Biofilmes/crescimento & desenvolvimento , Proteínas Ferro-Enxofre/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Filogenia , Domínios Proteicos/genética , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Vancomicina/uso terapêutico , Resistência a Vancomicina/genéticaRESUMO
Glutathione (GSH) is an abundantly produced low-molecular-weight (LMW) thiol in many organisms. However, a number of Gram-positive bacteria do not produce GSH, but instead produce bacillithiol (BSH) as one of the major LMW thiols. Similar to GSH, studies have found that BSH has various roles in the cell, including protection against hydrogen peroxide, hypochlorite and disulfide stress. BSH also participates in the detoxification of thiol-reactive antibiotics and the electrophilic metabolite methylglyoxal. Recently, a number of studies have highlighted additional roles for BSH in the processing of intracellular metals. Herein, we examine the potential functions of BSH in the biogenesis of Fe-S clusters, cytosolic metal buffering and the prevention of metal intoxication.
Assuntos
Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Metais/metabolismo , Cisteína/metabolismo , Glucosamina/metabolismo , Glutationa/metabolismo , Bactérias Gram-Positivas/metabolismo , Espaço Intracelular/metabolismo , Ferro/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase, verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus.
Assuntos
Proteínas de Bactérias/genética , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Proteínas Ferro-Enxofre/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Aconitato Hidratase/metabolismo , Apoproteínas/metabolismo , Cisteína/biossíntese , Cisteína/deficiência , Cisteína/fisiologia , Glucosamina/biossíntese , Glucosamina/deficiência , Glucosamina/fisiologia , Glutamato Sintase/metabolismo , Homeostase/genética , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxirredução , Fenótipo , Staphylococcus aureus/química , Enxofre/metabolismoRESUMO
The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron-sulfur (Fe-S) clusters, which are required for functional Fe-S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe-S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe-S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as an Fe-S cluster carrier, which aids in the maturation of Fe-S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non-incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe-S cluster metabolism as an attractive antimicrobial target.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Aconitato Hidratase/metabolismo , Animais , Dano ao DNA , Modelos Animais de Doenças , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/genética , Camundongos , Família Multigênica , Mutação , Neutrófilos/imunologia , Oxirredução , Ligação Proteica , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/genética , Enxofre/metabolismo , VirulênciaRESUMO
Staphylococcus aureus is an important human pathogen that employs a large repertoire of secreted virulence factors to promote disease pathogenesis. Many strains of S. aureus possess a plc gene that encodes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) capable of hydrolyzing PI and cleaving glycosyl-PI (GPI)-linked proteins from cell surfaces. Despite being secreted by virulent staphylococci, the contribution of PI-PLC to the capacity of S. aureus to cause disease remains undefined. Our goal in these studies was to understand PI-PLC in the context of S. aureus biology. Among a collection of genetically diverse clinical isolates of S. aureus, community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 secreted the most PI-PLC. Screening a collection of two-component system (TCS) mutants of S. aureus, we identified both the agr quorum-sensing system and the SrrAB TCS to be positive regulators of plc gene expression. Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator of plc. Furthermore, plc regulation was linked to oxidative stress both in vitro and in vivo in a SrrAB-dependent manner. A Δplc mutant in a CA-MRSA USA300 background exhibited a survival defect in human whole blood and in isolated neutrophils. However, the same mutant strain displayed no survival defect in murine models of infection or murine whole blood. Overall, these data identify potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis of S. aureus infections.
Assuntos
Neutrófilos/microbiologia , Fosfoinositídeo Fosfolipase C/fisiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Animais , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Lipase/metabolismo , Masculino , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Estresse Oxidativo/fisiologia , Fosfoinositídeo Fosfolipase C/sangue , Reação em Cadeia da Polimerase em Tempo Real , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Virulência/fisiologiaRESUMO
The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys(82)) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys(87) was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys(82). These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.
Assuntos
Ácidos Alcanossulfônicos/farmacologia , Compostos de Epóxi/metabolismo , Cetona Oxirredutases/metabolismo , Mesna/análogos & derivados , Cromatografia Líquida , Cetona Oxirredutases/antagonistas & inibidores , Espectrometria de Massas , NADP/metabolismoRESUMO
The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195-8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of "deviant" Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Metaloproteínas/química , Salmonella typhimurium/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Hidrólise , Cinética , Metaloproteínas/metabolismo , Ligação Proteica/fisiologiaRESUMO
Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.
Assuntos
Proteínas Arqueais , Proteínas Ferro-Enxofre , Methanococcales , Mathanococcus , Sulfolobus , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Teste de Complementação Genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/isolamento & purificação , Proteínas Ferro-Enxofre/metabolismo , Methanococcales/química , Methanococcales/genética , Methanococcales/metabolismo , Mathanococcus/química , Mathanococcus/genética , Mathanococcus/metabolismo , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sulfolobus/química , Sulfolobus/genética , Sulfolobus/metabolismoRESUMO
The metabolism of iron-sulfur ([Fe-S]) clusters requires a complex set of machinery that is still being defined. Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. ApbC is a 40.8 kDa homodimeric ATPase and as purified contains little iron and no acid-labile sulfide. An [Fe-S] cluster was reconstituted on ApbC, generating a protein that bound 2 mol of Fe and 2 mol of S (2-) per ApbC monomer and had a UV-visible absorption spectrum similar to known [4Fe-4S] cluster proteins. Holo-ApbC could rapidly and effectively activate Saccharomyces cerevisiae apo-isopropylmalate isolomerase (Leu1) in vitro, a process known to require the transfer of a [4Fe-4S] cluster. Maximum activation was achieved with 2 mol of ApbC per 1 mol of apo-Leu1. This article describes the first biochemical activity of ApbC in the context of [Fe-S] cluster metabolism. The data herein support a model in which ApbC coordinates an [4Fe-4S] cluster across its dimer interface and can transfer this cluster to an apoprotein acting as an [Fe-S] cluster scaffold protein, a function recently deduced for its eukaryotic homologues.
Assuntos
Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Ferro/química , Isomerases/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Enxofre/químicaRESUMO
The enzyme fructose-1,6-bisphosphatase (FBP) is key regulatory point in gluconeogenesis. Mutants of Salmonella enterica lacking purH accumulate 5-amino-4-imidazole carboxamide ribotide (AICAR) and are unable to utilize glycerol as sole carbon and energy sources. The work described here demonstrates this lack of growth is due to inhibition of FBP by AICAR. Mutant alleles of fbp that restore growth on glycerol encode proteins resistant to inhibition by AICAR and the allosteric regulator AMP. This is the first report of biochemical characterization of substitutions causing AMP resistance in a bacterial FBP. Inhibition of FBP activity by AICAR occurs at physiologically relevant concentrations and may represent a form of regulation of gluconeogenic flux in Salmonella enterica.