RESUMO
Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate.
Assuntos
Carbono , Ácido Cítrico , Regulação Bacteriana da Expressão Gênica , Homeostase , Ferro , Infecções Estafilocócicas , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Ferro/metabolismo , Carbono/metabolismo , Ácido Cítrico/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ciclo do Ácido Cítrico , Camundongos , Transdução de SinaisRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that predominantly causes nosocomial and community-acquired lung infections. As a member of ESKAPE pathogens, carbapenem-resistant P. aeruginosa (CRPA) compromises the limited therapeutic options, raising an urgent demand for the development of lead compounds against previously-unrecognized drug targets. Biotin is an important cofactor, of which the de novo synthesis is an attractive antimicrobial target in certain recalcitrant infections. Here we report genetic and biochemical definition of P. aeruginosa BioH (PA0502) that functions as a gatekeeper enzyme allowing the product pimeloyl-ACP to exit from fatty acid synthesis cycle and to enter the late stage of biotin synthesis pathway. In relative to Escherichia coli, P. aeruginosa physiologically requires 3-fold higher level of cytosolic biotin, which can be attributed to the occurrence of multiple biotinylated enzymes. The BioH protein enables the in vitro reconstitution of biotin synthesis. The repertoire of biotin abundance is assigned to different mouse tissues and/or organ contents, and the plasma biotin level of mouse is around 6-fold higher than that of human. Removal of bioH renders P. aeruginosa biotin auxotrophic and impairs its intra-phagosome persistence. Based on a model of CD-1 mice mimicking the human environment, lung challenge combined with systemic infection suggested that BioH is necessary for the full virulence of P. aeruginosa. As expected, the biotin synthesis inhibitor MAC13772 is capable of dampening the viability of CRPA. Notably, MAC13772 interferes the production of pyocyanin, an important virulence factor of P. aeruginosa. Our data expands our understanding of P. aeruginosa biotin synthesis relevant to bacterial infectivity. In particular, this study represents the first example of an extracellular pathogen P. aeruginosa that exploits biotin cofactor as a fitness determinant, raising the possibility of biotin synthesis as an anti-CRPA target.
Assuntos
Biotina , Infecções por Pseudomonas , Animais , Humanos , Camundongos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Biotina/química , Biotina/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMO
Nanolobatone A, featuring an unprecedented tricyclo[10.3.0.01,2 ]pentadecane carbon skeleton, along with four new polyoxygenated and four unusual endoperoxide-bridged casbane-type diterpenoids were isolated from the Hainan soft coral Sinularia nanolobata. The structures of the new compounds were established by extensive spectroscopic analysis, X-ray diffraction analysis, and time-dependent density functional theory/electronic circular dichroism calculations. A plausible biosynthetic pathway of new isolates was proposed. Bioassays revealed that nanolobatone A showed weak antibacterial activity against the Gram-positive bacteria Streptococcus pyogenes.
Assuntos
Antozoários , Diterpenos , Animais , Estrutura Molecular , Antozoários/química , Diterpenos/química , Dicroísmo Circular , Cristalografia por Raios XRESUMO
Novel type of Pd(II) complexes have been synthesized under operationally simple and convenient conditions and applied in the dynamic thermodynamic resolution of racemic N,C-unprotected α-amino acids. After rapid hydrolysis, these Pd(II) complexes produced the corresponding α-amino acids in satisfactory yields and enantioselectivities, accompanied by the recyclable proline-derived ligand. In addition, the method can be readily applied for S/R interconversion to obtain unnatural (R)-α-amino acids from readily available (S)-α-amino acids. Furthermore, biological assays showed that Pd(II) complexes (S,S)-3i and (S,S)-3m exhibited significant antibacterial activities similar to vancomycin, which may represent promising lead structures for further development of antibacterial agents.
Assuntos
Aminoácidos , Prolina , Prolina/química , Ligantes , Estereoisomerismo , Aminoácidos/química , Antibacterianos/farmacologia , TermodinâmicaRESUMO
An operationally simple and convenient resolution method via Cu(II) complexes was reported, efficiently providing valuable enantiopure N,C-unprotected α-amino acids. This protocol features synthetically attractive yields and a stereochemical outcome, using a recyclable Schiff base ligand and inexpensive easily accessible metal copper salts. These novel Cu(II) complexes can be obtained in an enantiopure state by means of column chromatography or recrystallization. Furthermore, all the Cu(II) complexes were evaluated for their antibacterial activities. Among them, complexes (S,2S)-3a, (S,2S)-3g, and (S,2S)-3o showed significant antibacterial activities against Staphylococcus aureus Mu50. Further biological evaluation indicated that they were effective against most of Gram-positive bacteria. It is the first study on the biological activities of transition metal complexes with this type of proline-derived Schiff base ligand.
Assuntos
Complexos de Coordenação , Bases de Schiff , Aminoácidos/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/farmacologia , Ligantes , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Prolina/farmacologia , Sais , Bases de Schiff/química , Bases de Schiff/farmacologiaRESUMO
Nine new isomalabaricane terpenoids (1-9) were isolated from the sponge Rhabdastrella globostellata of Ximao Island, together with 13 known ones (10-22). The structures were established by spectroscopic data interpretation and chemical calculations, as well as by comparison with spectroscopic data of known compounds. Notably, of the new isolates, hainanstelletin A (5) is the first representative of a nitrogenous isomalabaricane. The isolated compounds were evaluated against several cancer cell lines and two bacterial pathogens. In addition, moderate to strong antibacterial activities against Streptococcus pyogenes were also detected among geometric isomers 1, 2, and 10-12, with minimum inhibitory concentrations of 0.1-1.8 µg/mL.
Assuntos
Antineoplásicos , Poríferos , Triterpenos , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Terpenos/farmacologia , Triterpenos/químicaRESUMO
Four new sesquiterpene hydroquinones, xishaeleganins A-D (6-9), along with eleven known related ones (12 and 14-23) were isolated from the Xisha marine sponge Dactylospongia elegans (family Thorectida). Their structures were determined by extensive spectroscopic analysis, ECD calculations, and by comparison with the spectral data reported in the literature. Compounds 7, 15, 20, and 21 showed significant antibacterial activity against Staphylococcus aureus, with minimum inhibitory concentration values of 1.5, 2.9, 5.6, and 5.6 µg/mL, which are comparable with those obtained for the positive control vancomycin (MIC: 1.0 µg/mL).
Assuntos
Antibacterianos/farmacologia , Hidroquinonas/farmacologia , Poríferos/química , Sesquiterpenos/farmacologia , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Hidroquinonas/química , Hidroquinonas/isolamento & purificação , Testes de Sensibilidade Microbiana , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Análise Espectral , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologiaRESUMO
Intracellular protein degradation is essential for the survival of all organisms, but its role in interspecies interaction is unknown. Here, we show that the ClpXP protease of Pseudomonas aeruginosa suppresses its antimicrobial activity against Staphylococcus aureus, a common pathogen co-isolated with P. aeruginosa from polymicrobial human infections. Using proteomic, biochemical, and molecular genetic approaches, we found that this effect is due to the inhibitory effects of ClpXP on the quorum sensing (QS) of P. aeruginosa, mainly by degrading proteins (e.g., PhnA, PhnB, PqsR, and RhlI) which are critical for the production of QS signal molecules PQS and C4-HSL. We provide evidence that co-culturing with S. aureus induces a decrease in the activity of ClpXP in P. aeruginosa, an effect which was also achieved by the treatment of P. aeruginosa with N-acetylglucosamine (GlcNAc), a widespread chemical present on the surface of diverse cell types from bacteria to humans. These findings extend the range of biological events governed by proteolytic machinery to microbial community structure, thus also suggesting that a chemical-induced alteration of protein homeostasis is a mechanism for interspecies interactions.
Assuntos
Acetilglucosamina/farmacologia , Endopeptidase Clp/metabolismo , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/genética , Staphylococcus aureus/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Interações Microbianas , Mutação , Proteólise/efeitos dos fármacos , Proteômica , Proteostase , Infecções por Pseudomonas/microbiologia , Percepção de Quorum/efeitos dos fármacos , Infecções Estafilocócicas/microbiologiaRESUMO
Developing novel antibiotics is urgently needed with emergency of drug resistance. Vancomycin, the last resort for intractable Gram-positive bacterial infections, is ineffective against Gram-negative bacteria and vancomycin resistant bacteria. Herein, we report a series of novel vancomycin derivatives carrying LPS binding peptides, vancomycin-LPS binding peptide conjugates (VPCs). The LPS binding peptides were conjugated onto 4 sites of vancomycin via CuAAC or maleimide- sulfydryl addition, and the formed VPCs were screened against VISA/VRE and Gram-negative strains. VPCs exhibited enhanced activity against vancomycin resistant bacteria and obtained the activity against Gram-negative bacteria in vitro, providing a novel strategy for vancomycin modification and glycopeptide antibiotics synthesis.
Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Peptídeos/farmacologia , Vancomicina/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Lipopolissacarídeos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos/química , Relação Estrutura-Atividade , Vancomicina/químicaRESUMO
Over 60-year clinical use of vancomycin led to the emergence of vancomycin-resistant bacteria and threatened our health. To combat vancomycin-resistant strains, numerous vancomycin analogues were developed, such as Telavancin, Oritavancin and Dalbavancin. Extra structures embedded on C-terminus has been proved to be an effective strategy to promote antibacterial activity of vancomycin against vancomycin-resistant strains. Here, we reported a facile strategy, inspired by native chemical ligation, for vancomycin C-terminus functionalization and derivatization. The introduction of C-terminal hydrazide on vancomycin not only provided us an accessible method for C-terminus functionalization through carbonyl azide and thioester, also acted as an efficient site for vancomycin structure modifications. Based on hydrazide-vancomycin, we effectively conjugated cysteine and cysteine containing peptides onto vancomycin C-terminus, and two fluorescent FITC-vancomycin were prepared through Cys-Maleimide conjugation. Meanwhile, we introduced lipophilic structures onto vancomycin C-terminus via the hydrazide moiety. The obtained vancomycin derivatives were evaluated against both Gram-positive and negative bacteria strains.
Assuntos
Antibacterianos/farmacologia , Hidrazinas/farmacologia , Staphylococcus aureus Resistente à Vancomicina/efeitos dos fármacos , Vancomicina/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Humanos , Hidrazinas/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Vancomicina/síntese química , Vancomicina/químicaRESUMO
The fascinating and dangerous colored pathogens contain unique chemically pigmented molecules, which give varied and efficient assistance as virulence factors to the crucial reproduction and growth of microbes. Therefore, multiple novel strategies and inhibitors have been developed in recent years that target virulence factor pigments. However, despite the importance and significance of this topic, it has not yet been comprehensively reviewed. Moreover, research groups around the world have made successful progress against antibacterial infections by targeting pigment production, including our serial works on the discovery of CrtN inhibitors against staphyloxanthin production in Staphylococcus aureus. On the basis of the previous achievements and recent progress of our group in this field, this article will be the first comprehensive review of pigment inhibitors against colored pathogens, especially S. aureus infections, and this article includes design strategies, representative case studies, advantages, limitations, and perspectives to guide future research.
Assuntos
Anti-Infecciosos/farmacologia , Pigmentos Biológicos/antagonistas & inibidores , Fatores de Virulência/metabolismo , Animais , Anti-Infecciosos/química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Descoberta de Drogas , Reposicionamento de Medicamentos , Humanos , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismoRESUMO
Sepsis is a severe and complicated syndrome that is characterized by dysregulation of host inflammatory responses and organ failure, with high morbidity and mortality. The literature implies that autophagy is a crucial regulator of inflammation in sepsis. In this article, we report that autophagy-related protein 7 (Atg7) is involved in inflammasome activation in Pseudomonas aeruginosa abdominal infection. Following i.p. challenge with P. aeruginosa, atg7fl/fl mice showed impaired pathogen clearance, decreased survival, and widespread dissemination of bacteria into the blood and lung tissue compared with wild-type mice. The septic atg7fl/fl mice also exhibited elevated neutrophil infiltration and severe lung injury. Loss of Atg7 resulted in increased production of IL-1ß and pyroptosis, consistent with enhanced inflammasome activation. Furthermore, we demonstrated that P. aeruginosa flagellin is a chief trigger of inflammasome activation in the sepsis model. Collectively, our results provide insight into innate immunity and inflammasome activation in sepsis.
Assuntos
Proteína 7 Relacionada à Autofagia/imunologia , Inflamassomos/imunologia , Infecções por Pseudomonas/imunologia , Piroptose/imunologia , Sepse/imunologia , Animais , Proteína 7 Relacionada à Autofagia/deficiência , Modelos Animais de Doenças , Imunidade Inata/imunologia , Immunoblotting , Inflamassomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/imunologia , Sepse/metabolismoRESUMO
Increasing clinical significance of coagulase-negative staphylococci requires effective methods for species identification and genotyping. In this study, six housekeeping genes (femA, ftsZ, gap, pyrH, rpoB, and tuf) with extensive allelic polymorphisms were identified and evaluated to develop a comprehensive multilocus sequence typing (MLST) scheme. Selected primers were capable of amplification of the six loci from all of the 180 Staphylococcus strains belonging to 18 different species. Sequence analysis of each locus (44-63 alleles) revealed higher nucleotide diversity than 16S rRNA (28 alleles). Phylogenetic analysis of the concatenated sequences (3054 bp) of the six loci provided accurate species identification and highly discriminatory typing for all the strains. Multilocus allelic analysis of the 180 Staphylococcus strains generated 103 different sequence profiles, suggesting high genetic diversity of the strains. For example, 30 S. aureus, 37 S. epidermidis, 32 S. haemolyticus, and 14 S. hominis strains were typed into 15, 21, 11, and 10 sequence profiles, respectively. Compared with published MLST schemes that restrict on a few particular species, this new scheme both achieved similar discrimination for typing S. aureus, S. epidermidis, S. haemolyticus, and S. hominis and provided sufficient discriminatory power for typing additional opportunistic species, such as S. cohnii, S. capitis, and S. warneri. Importantly, the comprehensive MLST scheme for Staphylococcus strains provides a better genotyping tool for understanding the phylogeny of coagulase-positive Staphylococcus aureus strains.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Tipagem de Sequências Multilocus/métodos , RNA Ribossômico 16S/genética , Staphylococcus/classificação , Proteínas de Bactérias/genética , Coagulase/genética , Genótipo , Filogenia , Staphylococcus/isolamento & purificação , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificaçãoRESUMO
In the antibiotics arsenal, vancomycin is a last resort for the treatment of intractable infections. However, this situation is under threat because of the increasing appearance of vancomycin-resistant bacteria (VRB). Herein, we report a series of novel vancomycin derivatives carrying a sulfonium moiety. The sulfonium-vancomycin derivatives exhibited enhanced antibacterial activity against VRB both inâ vitro and inâ vivo. These derivatives also exhibited activity against some Gram-negative bacteria. The sulfonium modification enhanced the interaction of vancomycin with the bacterial cell membrane and disrupts membrane integrity. Furthermore, the inâ vivo pharmacokinetic profile, stability, and toxicity of these derivatives demonstrated good druggability of the sulfonium-vancomycin analogues. This work provides a promising strategy for combating drug-resistant bacterial infection, and advances the knowledge on sulfonium derivatives for structural optimization and drug development.
Assuntos
Farmacorresistência Bacteriana Múltipla/fisiologia , Compostos de Sulfônio/uso terapêutico , Vancomicina/uso terapêutico , Relação Estrutura-Atividade , Compostos de Sulfônio/farmacologia , Vancomicina/farmacologiaRESUMO
The surge of antibiotic resistance in Staphylococcus aureus has created a dire need for innovative anti-infective agents that attack new targets, to overcome resistance. In S. aureus, carotenoid pigment is an important virulence factor because it shields the bacterium from host oxidant killing. Here we show that naftifine, a US Food and Drug Administration (FDA)-approved antifungal drug, blocks biosynthesis of carotenoid pigment at nanomolar concentrations. This effect is mediated by competitive inhibition of S. aureus diapophytoene desaturase (CrtN), an essential enzyme for carotenoid pigment synthesis. We found that naftifine attenuated the virulence of a variety of clinical S. aureus isolates, including methicillin-resistant S. aureus (MRSA) strains, in mouse infection models. Specifically, we determined that naftifine is a lead compound for potent CrtN inhibitors. In sum, these findings reveal that naftifine could serve as a chemical probe to manipulate CrtN activity, providing proof of concept that CrtN is a druggable target against S. aureus infections.
Assuntos
Alilamina/análogos & derivados , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Alilamina/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Carotenoides/metabolismo , Desenho de Fármacos , Farmacorresistência Bacteriana , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Fatores de Virulência , Xantofilas/antagonistas & inibidores , Xantofilas/biossínteseRESUMO
LpxC inhibitors are new-type antibacterial agents developed in the last twenty years, mainly against Gram-negative bacteria infections. To develop novel LpxC inhibitors with good antibacterial activities and biological metabolism, we summarized the basic skeleton of reported LpxC inhibitors, designed and synthesized several series of compounds and tested their antibacterial activities against Escherichial coli and Pseudomonas aeruginosa in vitro. Structure-activity relationships have been discussed in this article. The metabolism stability of YDL-2, YDL-5, YDL-8, YDL-14, YDL-20-YDL-23 have been evaluated in liver microsomes, which indicated that the 2-amino isopropyl group may be a preferred structure than the 2-hydroxy ethyl group in the design of LpxC inhibitors.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Amidoidrolases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Sortase A (SrtA) anchors surface proteins to the cell wall and aids biofilm formation during infection, which functions as a key virulence factor of important Gram-positive pathogens, such as Staphylococcus aureus. At present researchers need a way in which to validate whether or not SrtA is a druggable target alternative to the conventional antibiotic targets in the mechanism. In this study, we performed a high-throughput screening and identified a new class of potential inhibitors of S. aureus SrtA, which are derived from natural products and contain the quinone skeleton. Compound 283 functions as an irreversible inhibitor that covalently alkylates the active site Cys184 of SrtA. NMR analysis confirms the direct interaction of the small-molecule inhibitor towards SrtA protein. The anchoring of protein A (SpA) to the cell wall and the biofilm formation are significantly attenuated when the S. aureus Newman strain is cultured in the presence of inhibitor. Our study indicates that compound 283 could be a potential hit for the development of new anti-virulence agents against S. aureus infections by covalently targeting SrtA.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Benzoquinonas/química , Inibidores de Cisteína Proteinase/química , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Transferência Ressonante de Energia de Fluorescência , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Peptídeos/análise , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologiaRESUMO
As a master redox-sensing MarR-family transcriptional regulator, AbfR participates in oxidative stress responses and virulence regulations in Staphylococcus epidermidis. Here, we present structural insights into the DNA-binding mechanism of AbfR in different oxidation states by determining the X-ray crystal structures of a reduced-AbfR/DNA complex, an overoxidized (Cys13-SO2H and Cys13-SO3H) AbfR/DNA, and 2-disulfide cross-linked AbfR dimer. Together with biochemical analyses, our results suggest that the redox regulation of AbfR-sensing displays two novel features: (i) the reversible disulfide modification, but not the irreversible overoxidation, significantly abolishes the DNA-binding ability of the AbfR repressor; (ii) either 1-disulfide cross-linked or 2-disulfide cross-linked AbfR dimer is biologically significant. The overoxidized species of AbfR, resembling the reduced AbfR in conformation and retaining the DNA-binding ability, does not exist in biologically significant concentrations, however. The 1-disulfide cross-linked modification endows AbfR with significantly weakened capability for DNA-binding. The 2-disulfide cross-linked AbfR adopts a very "open" conformation that is incompatible with DNA-binding. Overall, the concise oxidation chemistry of the redox-active cysteine allows AbfR to sense and respond to oxidative stress correctly and efficiently.
Assuntos
DNA/metabolismo , Staphylococcus epidermidis/metabolismo , Fatores de Transcrição/metabolismo , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Cristalografia por Raios X , DNA/química , Dissulfetos/química , Dissulfetos/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica , Fatores de Transcrição/químicaRESUMO
NalD was reported to be the secondary repressor of the MexAB-OprM multidrug efflux pump, the major system contributing to intrinsic multidrug resistance in Pseudomonas aeruginosa. Here, we show that novobiocin binds directly to NalD, which leads NalD to dissociate from the DNA promoter, and thus de-represses the expression of the MexAB-OprM pump. In addition, we have solved the crystal structure of NalD at a resolution of 2.90 Å. The structural alignment of NalD to its homologue TtgR reveals that the residues N129 and H167 in NalD are involved in its novobiocin-binding ability. We have confirmed the function of these two amino acids by EMSA and plate assay. The results presented here highlight the importance and diversity of regulatory mechanism in bacterial antibiotic resistance, and provide further insight for novel antimicrobial development.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Novobiocina/metabolismo , Pseudomonas aeruginosa/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/química , Cristalização , Cristalografia por Raios X , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Novobiocina/química , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Pseudomonas aeruginosa/metabolismo , Proteínas Repressoras/genéticaRESUMO
An effective metabolism is essential to all living organisms, including the important human pathogen Staphylococcus aureus. To establish successful infection, S. aureus must scavenge nutrients and coordinate its metabolism for proliferation. Meanwhile, it also must produce an array of virulence factors to interfere with host defenses. However, the ways in which S. aureus ties its metabolic state to its virulence regulation remain largely unknown. Here we show that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, binds to and activates the catabolite control protein E (CcpE) of S. aureus. Using structural and site-directed mutagenesis studies, we demonstrate that two arginine residues (Arg145 and Arg256) within the putative inducer-binding cavity of CcpE are important for its allosteric activation by citrate. Microarray analysis reveals that CcpE tunes the expression of 126 genes that comprise about 4.7% of the S. aureus genome. Intriguingly, although CcpE is a major positive regulator of the TCA-cycle activity, its regulon consists predominantly of genes involved in the pathogenesis of S. aureus. Moreover, inactivation of CcpE results in increased staphyloxanthin production, improved ability to acquire iron, increased resistance to whole-blood-mediated killing, and enhanced bacterial virulence in a mouse model of systemic infection. This study reveals CcpE as an important metabolic sensor that allows S. aureus to sense and adjust its metabolic state and subsequently to coordinate the expression of virulence factors and bacterial virulence.