RESUMEN
Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.
Asunto(s)
Bacillus subtilis , Regulación Bacteriana de la Expresión Génica , División del ARN , Estabilidad del ARN , ARN Bacteriano , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Conformación de Ácido Nucleico , Secuencia de BasesRESUMEN
Multi-resistant Staphylococcus aureus (S. aureus) infection is a significant global health concern owing to its high mortality and morbidity rates. Coagulase (Coa), a key enzyme that activates prothrombin to initiate host coagulation, has emerged as a promising target for anti-infective therapeutic approaches. This study identified sinigrin as a potent Coa inhibitor that significantly inhibited S. aureus-induced coagulation at concentration as low as 32 mg/L. Additionally, at a higher concentration of 128 mg/L, sinigrin disrupted the self-protection mechanism of S. aureus. Thermal shift and fluorescence-quenching assays confirmed the direct binding of sinigrin to the Coa protein. Molecular docking analysis predicted specific binding sites for sinigrin in the Coa molecule, and point mutation experiments highlighted the importance of Arg-187 and Asp-222 as critical binding sites for both Coa and sinigrin. In vivo studies demonstrated that the combination of sinigrin with oxacillin exhibited greater antibacterial efficacy than oxacillin alone in the treatment of S. aureus-induced pneumonia in mice. Furthermore, sinigrin was shown to reduce bacterial counts and inflammatory cytokine levels in the lung tissues of S. aureus-infected mice. In summary, sinigrin was shown to directly target Coa, resulting in the attenuation of S. aureus virulence, which suggests the potential of sinigrin as an adjuvant for future antimicrobial therapies.
Asunto(s)
Antibacterianos , Coagulasa , Simulación del Acoplamiento Molecular , Infecciones Estafilocócicas , Staphylococcus aureus , Coagulasa/metabolismo , Animales , Ratones , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/enzimología , Virulencia/efectos de los fármacos , Antibacterianos/farmacología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Modelos Animales de Enfermedad , Citocinas/metabolismo , Oxacilina/farmacología , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Pulmón/microbiología , Pulmón/patología , Femenino , Ratones Endogámicos BALB CRESUMEN
The bacterial pathogen Staphylococcus aureus employs a thick cell wall for protection against physical and chemical insults. This wall requires continuous maintenance to ensure strength and barrier integrity, but also to permit bacterial growth and division. The main cell wall component is peptidoglycan. Accordingly, the bacteria produce so-called peptidoglycan hydrolases (PGHs) that cleave glycan strands to facilitate growth, cell wall remodelling, separation of divided cells and release of exported proteins into the extracellular milieu. A special class of PGHs contains so-called 'cysteine, histidine-dependent amidohydrolase/peptidase' (CHAP) domains. In the present study, we profiled the roles of 11 CHAP PGHs encoded by the core genome of S. aureus USA300 LAC. Mutant strains lacking individual CHAP PGHs were analysed for growth, cell morphology, autolysis, and invasion and replication inside human lung epithelial cells. The results show that several investigated CHAP PGHs contribute to different extents to extracellular and intracellular growth and replication of S. aureus, septation of dividing cells, daughter cell separation once the division process is completed, autolysis and biofilm formation. In particular, the CHAP PGHs Sle1 and SAUSA300_2253 control intracellular staphylococcal replication and the resistance to ß-lactam antibiotics like oxacillin. This makes the S. aureus PGHs in general, and the Sle1 and SAUSA300_2253 proteins in particular, attractive targets for future prophylactic or therapeutic anti-staphylococcal interventions. Alternatively, these cell surface-exposed enzymes, or particular domains of these enzymes, could be applied in innovative anti-staphylococcal therapies.
Asunto(s)
Proteínas Bacterianas , Pared Celular , N-Acetil Muramoil-L-Alanina Amidasa , Staphylococcus aureus , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/genética , Humanos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Staphylococcus aureus/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pared Celular/metabolismo , Peptidoglicano/metabolismo , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Células Epiteliales/microbiologíaRESUMEN
Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyAâ¢membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.
Asunto(s)
Microscopía por Crioelectrón , Membrana Dobles de Lípidos , Liposomas , Staphylococcus aureus , Microscopía por Crioelectrón/métodos , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/química , Liposomas/química , Liposomas/metabolismo , Staphylococcus aureus/enzimología , Fosfolípidos/metabolismo , Fosfolípidos/química , Hidroliasas/química , Hidroliasas/metabolismo , Hidroliasas/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Modelos Moleculares , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Unión Proteica , Membrana Celular/metabolismoRESUMEN
Developing an effective Staphylococcus aureus (S. aureus) vaccine has been a challenging endeavor, as demonstrated by numerous failed clinical trials over the years. In this study, we formulated a vaccine containing a highly conserved moonlighting protein, the pyruvate dehydrogenase complex E2 subunit (PDHC), and showed that it induced strong protective immunity against epidemiologically relevant staphylococcal strains in various murine disease models. While antibody responses contributed to bacterial control, they were not essential for protective immunity in the bloodstream infection model. Conversely, vaccine-induced systemic immunity relied on γδ T cells. It has been suggested that prior S. aureus exposure may contribute to the reduction of vaccine efficacy. However, PDHC-induced protective immunity still facilitated bacterial clearance in mice previously exposed to S. aureus. Collectively, our findings indicate that PDHC is a promising serotype-independent vaccine candidate effective against both methicillin-sensitive and methicillin-resistant S. aureus isolates.
Asunto(s)
Infecciones Estafilocócicas , Vacunas Estafilocócicas , Staphylococcus aureus , Animales , Infecciones Estafilocócicas/prevención & control , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Ratones , Staphylococcus aureus/inmunología , Staphylococcus aureus/enzimología , Vacunas Estafilocócicas/inmunología , Complejo Piruvato Deshidrogenasa/metabolismo , Complejo Piruvato Deshidrogenasa/inmunología , Femenino , Anticuerpos Antibacterianos/inmunología , Modelos Animales de Enfermedad , Humanos , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Ratones Endogámicos C57BL , Staphylococcus aureus Resistente a Meticilina/inmunología , Piruvato Deshidrogenasa (Lipoamida)/inmunología , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Piruvato Deshidrogenasa (Lipoamida)/genéticaRESUMEN
Staphylococcus aureus (S. aureus) is a major bacterial infection in humans, leading to severe disease and causing death. The stagnation of antibiotic development in recent decades has made it difficult to combat drug-resistant infections. In this study, we performed an in silico structure-based drug screening (SBDS) targeting the S. aureus MurE (saMurE) enzyme involved in cell wall synthesis of S. aureus. saMurE is an enzyme that is essential for the survival of S. aureus but not present in humans. SBDS identified nine saMurE inhibitor candidates, Compounds 1-9, from a structural library of 154,118 compounds. Among them, Compound 2 showed strong antibacterial activity against Staphylococcus epidermidis (S. epidermidis) used as a model bacterium. Amino acid sequence homology between saMurE and S. epidermidis MurE is 87.4%, suggesting that Compound 2 has a similar inhibitory effect on S. aureus. Compound 2 showed an IC50 value of 301 nM for S. epidermidis in the dose-dependent growth inhibition assay. Molecular dynamics simulation showed that Compound 2 binds stably to both S. aureus MurD and S. aureus MurF, suggesting that it is a potential multi-pharmacological pharmacological inhibitor. The structural and bioactivity information of Compound 2, as well as its potential multiple-target activity, could contribute to developing new antimicrobial agents based on MurE inhibition.
Asunto(s)
Antibacterianos , Evaluación Preclínica de Medicamentos , Staphylococcus aureus , Antibacterianos/farmacología , Antibacterianos/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/enzimología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Simulación por Computador , Descubrimiento de Drogas , Relación Estructura-Actividad , Péptido Sintasas/antagonistas & inhibidores , Péptido Sintasas/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Humanos , Simulación de Dinámica MolecularRESUMEN
Iron is a crucial secondary metabolite for bacterial proliferation, but its bioavailability under infection conditions is limited by the low solubility of ferric ion and the host's ability to sequester iron by protein chelation. In these iron limiting conditions, bacteria produce and secrete low molecular weight ferric ion chelators, siderophores, to scavenge host iron. Iron bound siderophores are recognized by surface displayed receptors and internalized by active transport preceding the liberation of the iron payload by reduction or cleavage of the siderophore. The traditional paradigms surrounding the interactions between siderophores and their corresponding receptors have relied on canonical protein-ligand binding models that do not accurately reflect the conditions experienced by siderophore binding proteins (SBPs). Research by the Raymond group suggested that a ligand displacement model does not fully describe the role of SBPs in siderophore transport where the ferric ion can be shuttled between siderophore molecules during the transport process. This work inspired further research by the Wencewicz group, which demonstrated that the Staphylococcus aureus SBP FhuD2 can catalyze the transfer of iron from the biological iron source holo-transferrin to a SBP bound iron-free siderophore. The discovery of this ferrichelatase activity represents a novel mechanism of receptor mediated active transport which raises the question: is ferrichelatase activity a unique feature of FhuD2 or a previously unappreciated hallmark of SBPs? This chapter highlights a series of protocols for the general functional characterization of SBPs and methodologies to assay ferrichelatase activity with the hopes of providing the tools to answer this question.
Asunto(s)
Hierro , Sideróforos , Staphylococcus aureus , Sideróforos/metabolismo , Hierro/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/químicaRESUMEN
The hibernation-promoting factor (Hpf) in Staphylococcus aureus binds to 70S ribosomes and induces the formation of the 100S complex (70S dimer), leading to translational avoidance and occlusion of ribosomes from RNase R-mediated degradation. Here, we show that the 3'-5' exoribonuclease YhaM plays a previously unrecognized role in modulating ribosome stability. Unlike RNase R, which directly degrades the 16S rRNA of ribosomes in S. aureus cells lacking Hpf, YhaM destabilizes ribosomes by indirectly degrading the 3'-hpf mRNA that carries an intrinsic terminator. YhaM adopts an active hexameric assembly and robustly cleaves ssRNA in a manganese-dependent manner. In vivo, YhaM appears to be a low-processive enzyme, trimming the hpf mRNA by only 1 nucleotide. Deletion of yhaM delays cell growth. These findings substantiate the physiological significance of this cryptic enzyme and the protective role of Hpf in ribosome integrity, providing a mechanistic understanding of bacterial ribosome turnover.
Asunto(s)
Proteínas Bacterianas , Exorribonucleasas , ARN Mensajero , Proteínas Ribosómicas , Ribosomas , Staphylococcus aureus , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Ribosomas/metabolismo , Ribosomas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Estabilidad del ARN/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismoRESUMEN
Staphylococcus aureus (S. aureus) is abundant in nature and frequently leads to various health issues. Bacteriophages as obligate intracellular parasites of bacteria have the ability to specifically identify and infect S. aureus, causing bacterial lysis and the release of endogenous catalase (CAT). The released CAT triggers the conversion of H2O2 into O2 and H2O, resulting in a notable decrease in UV absorption at 570 nm and a concurrent surge in photocurrent. On the basis of this, a photoelectrochemical/colorimetric dual-mode biosensor for the detection of S. aureus was developed. In the photoelectric detection mode, the reactions involving endogenous enzymes occur directly in the solution, requiring only the simple drop-coating of TiO2@CdS onto the indium tin oxide (ITO) electrode surface. There was no need for immobilizing additional biomolecules, thereby significantly minimizing nonspecific adsorption and improving the biosensor's stability and reproducibility. For colorimetry, we utilized a cost-effective and operationally simple approach based on KI and starch. Remarkably, this photoelectrochemical/colorimetry exhibited a linear range of 102-109 CFU/mL for S. aureus, achieving detection limits of 7 and 10 CFU/mL, respectively. Herein, phage identification ensures the specific detection of live S. aureus, thereby effectively mitigating the potential for false signals. The dual-signal readout mode improves the detection accuracy and reliability. In conclusion, this present method offers numerous advantages, including simplicity, time-efficiency, cost-effectiveness, high specificity, and therefore excellent accuracy.
Asunto(s)
Técnicas Biosensibles , Catalasa , Colorimetría , Técnicas Electroquímicas , Staphylococcus aureus , Staphylococcus aureus/enzimología , Staphylococcus aureus/aislamiento & purificación , Catalasa/metabolismo , Catalasa/química , Técnicas Biosensibles/métodos , Colorimetría/métodos , Compuestos de Estaño/química , Compuestos de Cadmio/química , Titanio/química , Sulfuros/química , Procesos Fotoquímicos , Electrodos , Límite de DetecciónRESUMEN
Enzymatic site-specific bioconjugation techniques, in particular sortase-mediated ligation, are increasingly used to generate conjugated proteins for a wide array of applications. Extension of the utility and practicality of sortagging for diverse purposes is critically dependent on further improvement of the efficiency of sortagging reactions with a wider structural variety of substrates. We present a comprehensive comparative mass spectrometry screening study of synthetic nonpeptidic incoming amine nucleophile substrates of Staphylococcus aureus Sortase A enzyme. We have identified the optimal structural motifs among the chemically diverse set of 452 model primary and secondary amine-containing sortagging substrates, and we demonstrate the utility of representative amine linkers for efficient C-terminal biotinylation of nanobodies.
Asunto(s)
Aminas , Aminoaciltransferasas , Proteínas Bacterianas , Cisteína Endopeptidasas , Staphylococcus aureus , Aminoaciltransferasas/metabolismo , Aminas/química , Staphylococcus aureus/enzimología , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/química , Proteínas Bacterianas/química , Biotinilación , Especificidad por Sustrato , Anticuerpos de Dominio Único/química , Espectrometría de MasasRESUMEN
Staphylococcus aureus signal peptidase IB (SpsB) is an essential enzyme for protein secretion. While inhibition of its activity by small molecules is a well-precedented mechanism to kill bacteria, the mode of activation is however less understood. We here investigate the activation mechanism of a recently introduced activator, the antibiotic compound PK150, and demonstrate by combined experimental and Molecular Dynamics (MD) simulation studies a unique principle of enzyme stimulation. Mass spectrometric studies with an affinity-based probe of PK150 unravel the binding site of PK150 in SpsB which is used as a starting point for MD simulations. Our model shows the localization of the molecule in an allosteric pocket next to the active site which shields the catalytic dyad from excess water that destabilizes the catalytic geometry. This mechanism is validated by the placement of mutations aligning the binding pocket of PK150. While the mutants retain turnover of the SpsB substrate, no stimulation of activity is observed upon PK150 addition. Overall, our study elucidates a previously little investigated mechanism of enzyme activation and serves as a starting point for the development of future enzyme activators.
Asunto(s)
Proteínas Bacterianas , Simulación de Dinámica Molecular , Serina Endopeptidasas , Staphylococcus aureus , Staphylococcus aureus/enzimología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Activación Enzimática , Sitios de Unión , Antibacterianos/farmacología , Dominio CatalíticoRESUMEN
Staphylococcus aureus, a Gram-positive bacterium, is an opportunistic pathogen and one of the most frequent causes for community acquired and nosocomial infections that has become a major public health threat due to the increased incidence of its drug resistance. Although being a prominent pathogen, its energetic metabolism is still underexplored, and its respiratory enzymes have been escaping attention. S. aureus can adapt to different environmental conditions by performing both aerobic and anaerobic respirations, which is particularly important as it frequently colonizes niches with different oxygen concentrations. This adaptability is derived from the composition of its respiratory chain, specifically from the presence of terminal electron acceptor reductases. The plasticity of S. aureus energy metabolism is enlarged by the ten quinone reductases encoded in its genome, eight of them being monotopic proteins. The role of these proteins is critical as they connect the different catabolic pathways to the respiratory chain. In this work, we identify, describe, and revise the monotopic quinone reductases present in S. aureus, providing an integrated view of its respiratory chain.
Asunto(s)
Staphylococcus aureus , Staphylococcus aureus/enzimología , Quinona Reductasas/metabolismo , Quinona Reductasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Transporte de Electrón , Metabolismo EnergéticoRESUMEN
BACKGROUND: Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target. OBJECTIVE: In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment. METHODS: SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied. RESULTS: SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ. CONCLUSION: The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer.
Asunto(s)
Proteínas Bacterianas , Staphylococcus aureus , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Solubilidad , Expresión Génica , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivadosRESUMEN
Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently. The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow S. aureus to live and survive. Despite its prominence as a pathogen, the energy metabolism of S. aureus remains underexplored, with its respiratory enzymes often escaping thorough investigation. S. aureus bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of S. aureus to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.
Asunto(s)
Proteínas Bacterianas , NAD , Quinona Reductasas , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Staphylococcus aureus/metabolismo , NAD/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Quinona Reductasas/metabolismo , Quinona Reductasas/genética , NADP/metabolismo , Metabolismo Energético , Oxidación-ReducciónRESUMEN
Staphylococcus aureus secretes an array of small proteins that inhibit key enzyme-catalyzed reactions necessary for proper function of the human innate immune system. Among these, the Staphylococcal Peroxidase Inhibitor, SPIN, blocks the activity of myeloperoxidase (MPO) and thereby disrupts the HOCl-generating system of neutrophils. Previous studies on S. aureus SPIN have shown that it relies on a C-terminal α-helical bundle domain to mediate initial binding to MPO, but requires a disordered N-terminal region to fold into a ß-hairpin conformation to inhibit MPO activity. To further investigate the structure/function relationship of SPIN, we introduced two cysteine residues into its N-terminal region to trap SPIN in its MPO-bound conformation and characterized the modified protein, which we refer to here as SPIN-CYS. Although control experiments confirmed the presence of the disulfide bond in SPIN-CYS, solution structure determination revealed that the N-terminal region of SPIN-CYS adopted a physically constrained series of lariat-like structures rather than a well-defined ß-hairpin. Nevertheless, SPIN-CYS exhibited a gain in inhibitory potency against human MPO when compared to wild-type SPIN. This gain of function persisted even in the presence of deleterious mutations within the C-terminal α-helical bundle domain. Surface plasmon resonance studies showed that the gain in potency arose through an increase in apparent affinity of SPIN-CYS for MPO, which was driven primarily by an increased association rate with MPO when compared to wild-type SPIN. Together, this work provides new information on the coupled binding and folding events required to manifest biological activity of this unusual MPO inhibitor.
Asunto(s)
Disulfuros , Peroxidasa , Staphylococcus aureus , Staphylococcus aureus/enzimología , Disulfuros/química , Disulfuros/metabolismo , Peroxidasa/química , Peroxidasa/antagonistas & inhibidores , Peroxidasa/metabolismo , Humanos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Dominios Proteicos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cisteína/química , Cisteína/metabolismo , Modelos MolecularesRESUMEN
Pathogenic bacteria employ complex systems to cope with metal ion shortage conditions and propagate in the host. IsrR is a regulatory RNA (sRNA) whose activity is decisive for optimum Staphylococcus aureus fitness upon iron starvation and for full virulence. IsrR down-regulates several genes encoding iron-containing enzymes to spare iron for essential processes. Here, we report that IsrR regulates the tricarboxylic acid (TCA) cycle by controlling aconitase (CitB), an iron-sulfur cluster-containing enzyme, and its transcriptional regulator, CcpE. This IsrR-dependent dual-regulatory mechanism provides an RNA-driven feedforward loop, underscoring the tight control required to prevent aconitase expression. Beyond its canonical enzymatic role, aconitase becomes an RNA-binding protein with regulatory activity in iron-deprived conditions, a feature that is conserved in S. aureus. Aconitase not only negatively regulates its own expression, but also impacts the enzymes involved in both its substrate supply and product utilization. This moonlighting activity concurrently upregulates pyruvate carboxylase expression, allowing it to compensate for the TCA cycle deficiency associated with iron scarcity. These results highlight the cascade of complex posttranscriptional regulations controlling S. aureus central metabolism in response to iron deficiency.
Asunto(s)
Aconitato Hidratasa , Proteínas Bacterianas , Ciclo del Ácido Cítrico , Regulación Bacteriana de la Expresión Génica , Staphylococcus aureus , Aconitato Hidratasa/metabolismo , Aconitato Hidratasa/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hierro/metabolismo , Deficiencias de Hierro , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN Pequeño no Traducido/metabolismo , ARN Pequeño no Traducido/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/enzimologíaRESUMEN
Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections.
Asunto(s)
Proteínas Bacterianas , Ligasas de Carbono-Nitrógeno , Staphylococcus aureus , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/aislamiento & purificación , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/biosíntesis , Expresión Génica , Simulación del Acoplamiento Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesisRESUMEN
Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σA controls housekeeping functions, S. aureus σB regulates virulence, biofilm formation, persistence, cell internalization, membrane transport, and antimicrobial resistance. Besides the sequence difference, the spacers between the -35 element and -10 element of σB regulated promoters are shorter than those of σA regulated promoters. Therefore, how σB recognizes and initiates transcription from target promoters can not be inferred from that of the well studied σ. Here, we report the cryo-EM structures of S. aureus RNAP-promoter open complexes comprising σA and σB, respectively. Structural analyses, in combination with biochemical experiments, reveal the structural basis for the promoter specificity of S. aureus transcription. Although the -10 element of σA regulated promoters is recognized by domain σA2 as single-stranded DNA, the -10 element of σB regulated promoters is co-recognized by domains σB2 and σB3 as double-stranded DNA, accounting for the short spacers of σB regulated promoters. S. aureus RNAP is a validated target of antibiotics, and our structures pave the way for rational drug design targeting S. aureus RNAP.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN , Regiones Promotoras Genéticas , Factor sigma , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/química , Factor sigma/metabolismo , Factor sigma/genética , Factor sigma/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Transcripción Genética , Unión ProteicaRESUMEN
The nanozyme-linked aptamer-sorbent assay (NLASA) is a rapid way to screen and characterize aptamer binding to targets. In this paper, a MnO2@AuNPs@aptamer (Apt) based NLASA coupled with colorimetric-SERS dual-mode for Staphylococcus aureus (S. aureus) detection is presented. Cu,Fe-CDs were used as the reducing agent to synthesize MnO2 and gold nanoparticles (AuNPs). Then, they were fabricated to obtain MnO2@AuNPs with oxidase (OXD)-like and SERS activities. The S. aureus aptamer was conjugated to MnO2@AuNPs and enhanced the OXD-like activity, which realized the specific capture of S. aureus in food matrices. In addition, S. aureus improves the oxidation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) but inhibits 3,3',5,5'-tetramethylbenzidine (TMB) to generate Raman-active oxTMB with MnO2@AuNPs@Apt. This sensor was used for detections of S. aureus in a concentration ranged from 101 to 107 CFU/mL with a detection limit of 0.926 CFU/mL (colorimetric) and 1.561 CFU/mL (SERS), and the recovery is 85%-105% in real samples.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Colorimetría , Oro , Compuestos de Manganeso , Nanopartículas del Metal , Óxidos , Oxidorreductasas , Staphylococcus aureus , Staphylococcus aureus/enzimología , Colorimetría/métodos , Oro/química , Compuestos de Manganeso/química , Óxidos/química , Nanopartículas del Metal/química , Aptámeros de Nucleótidos/química , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Espectrometría Raman/métodosRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.