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1.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502568

RESUMO

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (Km = 0.23 +/- 0.013 x 10-3 M; Vmax = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na+ ions inhibit the enzyme while increasing concentrations of K+ ions were stimulatory. In case of divalent cations, it was found that Mg2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn2+, Mn2+, Fe2+, Cu2+, Ni2+ and Ca2+) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg2+ and Cr2+. The circular dichroism data indicate that other than Hg2+, Ni2+ and to a certain extent Cu2+, none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.


Assuntos
Estabilidade Enzimática/efeitos dos fármacos , Metais/farmacologia , Fosfopiruvato Hidratase/genética , Conformação Proteica/efeitos dos fármacos , Catálise/efeitos dos fármacos , Dicroísmo Circular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Íons/química , Íons/farmacologia , Metais/química , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/isolamento & purificação , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade
2.
J Recept Signal Transduct Res ; 39(3): 283-293, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31538846

RESUMO

A new series of benzothiazol-2-ylcarbamodithioate functional compounds 5a-f has been designed, synthesized and characterized by spectral data. These compounds were screened for their in vitro antibacterial activity against strains of Staphylococcus aureus (NCIM 5021, NCIM 5022 and methicillin-resistant isolate 43300), Bacillus subtilis (NCIM 2545), Escherichia coli (NCIM 2567), Klebsiella pneumoniae (NCIM 2706) and Psudomonas aeruginosa (NCIM 2036). Compounds 5a and 5d exhibited significant activity against all the tested bacterial strains. Specifically, compounds 5a and 5d showed potent activity against K. pneumoniae (NCIM 2706), while compound 5a also displayed potent activity against S. aureus (NCIM 5021). Compound 5d showed minimum IC50 value of 13.37 µM against S. aureus MurD enzyme. Further, the binding interactions of compounds 5a-f in the catalytic pocket have been investigated using the extra-precision molecular docking and binding free energy calculation by MM-GBSA approach. A 30 ns molecular dynamics simulation of 5d/modeled S. aureus MurD enzyme was performed to determine the stability of the predicted binding conformation.


Assuntos
Benzotiazóis/síntese química , Benzotiazóis/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Sintases/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Benzotiazóis/química , Benzotiazóis/farmacocinética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Peptídeo Sintases/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Termodinâmica
3.
J Microbiol Biotechnol ; 29(10): 1603-1606, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31474099

RESUMO

Sortase A (SrtA), a type of transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall, is important in the virulence of gram-positive bacteria. Three compounds were isolated from marine-derived Streptomyces sp. MBTH32 using various chromatography techniques. The structures of these compounds were determined based on spectroscopic data and comparisons with previously reported data. Among the metabolites tested, lumichrome showed strong inhibitory activity against Staphylococcus aureus SrtA without affecting cell viability. The results of cell clumping activity assessment suggest the potential for using this compound to treat S. aureus infection by inhibiting SrtA activity.


Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibrinogênio/metabolismo , Staphylococcus aureus/patogenicidade , Streptomyces/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Flavinas/química , Flavinas/isolamento & purificação , Flavinas/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Mutação , Staphylococcus aureus/enzimologia , Streptomyces/metabolismo , Virulência/efeitos dos fármacos
4.
Molecules ; 24(17)2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466322

RESUMO

A series of Schiff bases 14-25 were designed and synthesized for evaluation of their antibacterial properties against multi-drug resistant bacteria (MDRB). The antibacterial activities of Schiff bases 14-25 showed that most of the synthesized compounds displayed a significant antibacterial activity. Assessment of in silico ADMET properties (absorption, distribution, metabolism, excretion and toxicity) of Schiff bases illustrates that all derivatives showed agreement to the Lipinski's rule of five. Further enzymatic assay aided by molecular docking study demonstrated that compound 18 is a potent inhibitor of staphylococcus aureus DNA gyrase and dihydrofolate reductase kinases. This study could be valuable in the discovery of new potent antimicrobial agents.


Assuntos
Antibacterianos/síntese química , Pirazóis/química , Bases de Schiff/síntese química , Staphylococcus aureus/enzimologia , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Simulação por Computador , DNA Girase/metabolismo , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Bases de Schiff/química , Bases de Schiff/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/metabolismo , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia
5.
Biochimie ; 165: 235-244, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422053

RESUMO

The 2H phosphoesterase superfamily (2H family) proteins are widely conserved among organisms. The 2H family is classified into several subgroups, including YjcG-like proteins whose enzymatic activity has not been reported. In the present study, we found that two YjcG-like proteins (Staphylococcus aureus SA0873 and Bacillus subtilis YjcG) have 2'-CPDase activity that hydrolyzes a 2',3'-cyclic nucleotide, thereby producing a nucleotide with a 3'-phosphate. The SA0873 protein selectively hydrolyzes a 2',3'-cyclic nucleotide with a purine base. Four SA0873 mutant proteins (H34A, T36A, H115A, and T117A), in which alanine was substituted for amino acid residues in the HxT/Sx motifs that are conserved in the 2H family, abolished the 2'-CPDase activity. Comparison of three-dimensional structures between the YjcG-like proteins with 2'-CPDase activity and another 2H family subgroup, LigT/2'-5' RNA ligase-like proteins with 3'-CPDase activity, revealed that the orientation of the substrate binding pocket is reversed between the two groups. Our findings revealed that YjcG-like proteins not only have a substrate-binding pocket different from that of LigT/2'-5' RNA ligase-like proteins, but they also have 2'-CPDase activity.


Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/química , Bacillus subtilis/enzimologia , Staphylococcus aureus/enzimologia , Domínio Catalítico , Modelos Moleculares , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
6.
mSphere ; 4(4)2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292227

RESUMO

The virulence of the human pathogen Staphylococcus aureus is supported by many heme-dependent proteins, including key enzymes of cellular respiration. Therefore, synthesis of heme is a critical component of staphylococcal physiology. S. aureus generates heme via the coproporphyrin-dependent pathway, conserved across members of the Firmicutes and Actinobacteria In this work, we genetically investigate the oxidation of coproporphyrinogen to coproporphyrin in this heme synthesis pathway. The coproporphyrinogen III oxidase CgoX has previously been identified as the oxygen-dependent enzyme responsible for this conversion under aerobic conditions. However, because S. aureus uses heme during anaerobic nitrate respiration, we hypothesized that coproporphyrin production is able to proceed in the absence of oxygen. Therefore, we tested the contribution to anaerobic heme synthesis of CgoX and two other proteins previously identified as potential oxygen-independent coproporphyrinogen dehydrogenases, NWMN_1486 and NWMN_1636. We have found that CgoX alone is responsible for aerobic and anaerobic coproporphyrin synthesis from coproporphyrinogen and is required for aerobic and anaerobic heme-dependent growth. This work provides an explanation for how S. aureus heme synthesis proceeds under both aerobic and anaerobic conditions.IMPORTANCE Heme is a critical molecule required for aerobic and anaerobic respiration by organisms across kingdoms. The human pathogen Staphylococcus aureus has served as a model organism for the study of heme synthesis and heme-dependent physiology and, like many species of the phyla Firmicutes and Actinobacteria, generates heme through a coproporphyrin intermediate. A critical step in terminal heme synthesis is the production of coproporphyrin by the CgoX enzyme, which was presumed to be oxygen dependent. However, S. aureus also requires heme during anaerobic growth; therefore, the synthesis of coproporphyrin by an oxygen-independent mechanism is required. Here, we identify CgoX as the enzyme performing the oxygen-dependent and -independent synthesis of coproporphyrin from coproporphyrinogen, resolving a key outstanding question in the coproporphyrin-dependent heme synthesis pathway.


Assuntos
Coproporfirinogênio Oxidase/metabolismo , Heme/biossíntese , Staphylococcus aureus/enzimologia , Aerobiose , Anaerobiose , Coproporfirinogênio Oxidase/genética , Coproporfirinogênios/metabolismo , Oxirredução , Staphylococcus aureus/genética , Virulência
7.
Adv Mater ; 31(33): e1902462, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31265196

RESUMO

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though "smart" platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user-specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site-specifically modified proteins from hydrogels following Boolean logic. A sortase-mediated transpeptidation reaction is used to generate recombinant proteins C-terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli-labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two-input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli-sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Materiais Biocompatíveis/química , Cisteína Endopeptidases/química , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/química , Proteínas Recombinantes/química , Aminoaciltransferases/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Cisteína Endopeptidases/administração & dosagem , Dissulfetos/química , Liberação Controlada de Fármacos , Humanos , Luz , Oxirredução , Peptídeos/química , Fotólise , Polietilenoglicóis/química , Proteínas Recombinantes/administração & dosagem , Staphylococcus aureus/enzimologia
8.
J Agric Food Chem ; 67(30): 8382-8392, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31271032

RESUMO

Staphylococcus aureus is a common pathogen that can cause life-threatening infections. Treatment of antibiotic-resistant S. aureus infection needs effective antibacterial agents. Thymol, a generally recognized safe natural compound, has potential as an alternative to treat S. aureus infections. However, the targets and mechanisms of action of thymol were not fully understood. Bioinformatics analysis showed that IolS, a predicted aldo-keto reductase (AKR) in S. aureus, could be a potential target of thymol. Isothermal titration calorimetry (ITC) analysis demonstrated that thymol directly binds IolS and amino acid residues (Y30 and L33) are essential for such binding. Deletion of IolS or mutation of Y30A and L33A reduced the bactericidal activity of thymol at the concentration of 200 µg/mL, suggesting that thymol mediates bactericidal activity via binding with IolS. Biochemical analysis showed that addition of thymol significantly increased AKR activity of IolS from 1.6 ± 0.1 to 2.4 ± 0.2 U (p < 0.05). The content of NADPH within S. aureus cells decreased significantly from 105 ± 5 to 72 ± 3 pmol/108 cells (p < 0.05) following thymol treatment at the concentration of 200 µg/mL. Importantly, addition of NADPH could alleviate the bactericidal effect of thymol on S. aureus, indicating that the depletion of NADPH is responsible for thymol-mediated bactericidal activity. Overall, these results demonstrated that thymol could directly bind IolS and increase its AKR activity, leading to the depletion of NADPH and bactericidal effect. AKR activity of IolS could be a promising target for the development of new antimicrobials.


Assuntos
Aldo-Ceto Redutases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , NADP/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Timol/farmacologia , Aldo-Ceto Redutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
9.
Talanta ; 204: 693-699, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357354

RESUMO

Micrococcal nuclease (MNase) is a nonspecific endo-exonuclease that digests single-stranded/double-stranded DNA and RNA. The existence of MNase can serves as an important diagnostic biomarker of Staphylococcus aureus (S. aureus) infection. However, most of the substrates in MNase-based sensors are single-stranded DNA, which could also be digested by exonuclease I or S1 nuclease and interfere the MNase detection. In this work, we developed a highly selective fluorescent method for MNase detection using a specific dsDNA and nucleic acid dye SYBR Green I (SGI) as the indicator. After rational design, an AT-rich dsDNA with 3' protruding termini was screened as the high-specific substrate of MNase assay and efficient enhancer of SGI. The AT-rich dsDNA substrate can resist the digestion of other exonuclease and greatly enhance the fluorescence of SGI. This high-specific substrate-based probe can detect MNase in buffer as well as biological sample with highly selectivity. Moreover, this method was also applied to monitor the MNase secreted by S. aureus. Thus, the proposed MNase-based assay has a strong potential to identify S. aureus in food safety and microbial infection due to its excellent analytical sensitivity and high selectivity.


Assuntos
DNA/química , Ensaios Enzimáticos/métodos , Nuclease do Micrococo/sangue , Staphylococcus aureus/isolamento & purificação , Biomarcadores/sangue , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Compostos Orgânicos/química , Espectrometria de Fluorescência , Staphylococcus aureus/enzimologia
10.
J Dairy Sci ; 102(8): 6923-6927, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178176

RESUMO

Reports of ß-lactam-resistant Staphylococcus aureus in artisanal goat cheese are increasing, and this phenomenon is relevant to public health. The objective of the present study was to determine the prevalence of S. aureus strains carrying the blaZ and mecA resistance genes, as well as the genes encoding the staphylococcal enterotoxins SEA, SEB, SEC, SED, SEE, and TSST-1 in artisanal coalho cheese made from goat milk produced in northeastern Brazil. We used biochemical and molecular tests to characterize 54 S. aureus isolates found in artisanal coalho cheese collected from commercial establishments producing animal products in 11 municipalities of Pernambuco State, Brazil. A PCR analysis revealed that 42.6% (23/54) of the isolates were positive for the blaZ gene, and 7.4% (4/54) were resistant to methicillin by phenotypic testing. We did not detect mecA or any genes encoding enterotoxins. The presence of S. aureus carriers of the blaZ gene and the identification of methicillin-resistant S. aureus strains are of concern for the health of consumers of this type of cheese.


Assuntos
Queijo/microbiologia , Resistência a Meticilina/genética , Leite/microbiologia , Staphylococcus aureus/fisiologia , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Brasil , Feminino , Cabras , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/fisiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética
11.
Analyst ; 144(13): 3999-4005, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31172144

RESUMO

Micrococcal nuclease (MNase) is an extracellular endonuclease of Staphylococcus aureus (S. aureus). It digests single stranded nucleic acid. The presence of MNase is the gold standard to identify S. aureus and its content. The present study reports the ultrahigh sensitive and selective fluorescence platform for MNase detection, designed and developed based on the surface energy transfer mechanism. A "proof of concept" is being developed based on monoclonal antibody-conjugated quantum dots (mAb-QDs), wherein mAb-QDs act as donors and graphene oxide (GO) acts as an acceptor. mAb-QDs in close proximity to GO undergo adsorption due to weak affinity between them and this results in fluorescence quenching by the transfer of surface energy from mAb-QDs to GO. During sensing, a much stronger affinity of mAb-QDs towards MNase inhibits the energy transfer to GO and this allows the regaining of fluorescence. Immobilized mAb-QDs on nitrocellulose membrane strips were fabricated and tested for "ON-OFF-ON" sensing of MNase. The limit of detection for fluorescence based assay and strips is found to be 0.3 ng mL-1 and 0.5 ng mL-1, respectively. The developed strips were applied on real samples for the detection of S. aureus.


Assuntos
Corantes Fluorescentes/química , Grafite/química , Nuclease do Micrococo/análise , Pontos Quânticos/química , Staphylococcus aureus/química , Anticorpos Monoclonais/química , Técnicas Biossensoriais/métodos , Colódio/química , Transferência Ressonante de Energia de Fluorescência/métodos , Limite de Detecção , Membranas Artificiais , Nanocompostos , Estudo de Prova de Conceito , Sensibilidade e Especificidade , Staphylococcus aureus/enzimologia
12.
J Med Microbiol ; 68(6): 952-956, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31107204

RESUMO

Exploiting the immunosuppressive, analgesic and highly addictive properties of morphine could increase the success of a bacterial pathogen. Therefore, we performed sequence similarity searches for two morphine biosynthesis demethylases in bacteria. For thebaine 6-O-demethylase and codeine O-demethylase, we found strong alignments to three (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) of the six ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) that are commonly associated with drug resistance and nosocomial infections. Expression of the aligned sequence found in P. aeruginosa (NP_252880.1/PA4191) is upregulated in isolates obtained from cystic fibrosis patients. Our findings provide putative mechanistic targets for understanding the role of morphine in pathogenicity.


Assuntos
Acinetobacter baumannii/enzimologia , Infecção Hospitalar/microbiologia , Enterobacter/enzimologia , Klebsiella pneumoniae/enzimologia , Oxirredutases O-Desmetilantes/genética , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Acinetobacter baumannii/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Codeína/metabolismo , Enterobacter/genética , Humanos , Klebsiella pneumoniae/genética , Derivados da Morfina/metabolismo , Alcaloides Opiáceos/administração & dosagem , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Staphylococcus aureus/genética , Tebaína/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-31041195

RESUMO

The two coagulases, von Willebrand factor binding protein (vWbp) and Coagulase (Coa), are critical virulence factors in several animal models of invasive Staphylococcus aureus (S. aureus) infections. These proteins are part of an intricate system of proteins that S. aureus uses to assemble a fibrinogen (Fg)/fibrin protective shield surrounding itself. This shield allows the microorganism to evade clearance by the host phagocytic cells. The coagulases can non-proteolytically activate the zymogen prothrombin to convert Fg to fibrin and promote the Fg/fibrin shield formation. The coagulases also bind directly to Fg and the interaction between Coa and Fg has been previously characterized in some detail. However, the mechanism(s) by which vWbp interacts with Fg remains unclear. Here, we show that vWbp and Coa have distinct interactions with Fg, despite being structurally similar. Coa binds with a significantly higher affinity to soluble Fg than to Fg coated on a plastic surface, whereas vWbp demonstrates no preference between the two forms of Fg. The two coagulases appear to target different sites on Fg, as they do not compete with each other in binding to Fg. Similar to Coa, both the N- and C-terminal halves of vWbp (vWbp-N, vWbp-C, respectively) harbor Fg-binding activities. The higher affinity Fg-binding activity resides in vWbp-N; whereas, the C-terminal region of Coa encompasses the major Fg-binding activity. Peptides constituting the previously identified Coa/Efb1 Fg-binding motif fail to inhibit vWbp-C from binding to Fg, indicating that vWbp-C lacks a functional homolog to this motif. Interestingly, the N-terminal prothrombin-binding domains of both coagulases recognize the Fg ß-chain, but they appear to interact with different sequence motifs in the host protein. Collectively, our data provide insight into the complex interactions between Fg and the S. aureus coagulases.


Assuntos
Proteínas de Transporte/metabolismo , Coagulase/metabolismo , Fibrinogênio/metabolismo , Staphylococcus aureus/enzimologia , Fatores de Virulência/metabolismo , Sítios de Ligação , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas
14.
Future Med Chem ; 11(9): 935-945, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31140866

RESUMO

Aim: Novel bacterial topoisomerase inhibitors (NBTIs) are a promising class of bacterial topoisomerase II inhibitors that are gaining more and more importance mainly because of their excellent antibacterial activity, as well as their lack of cross-resistance to quinolones. Results: Described here is the synthesis and biological evaluation of a tiny series of new virtually assembled NBTIs containing synthetically feasible right-hand side fragments capable of binding the GyrA subunit of the bacterial DNA gyrase-DNA complex. Conclusion: NBTI variants with incorporated 1-phenylpyrazole right-hand side moiety show suitable antibacterial activity against Gram-positive Staphylococcus aureus, with confirmed selectivity over the human topoisomerase IIα enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , DNA Bacteriano/metabolismo , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Cicloexanóis/química , Cicloexanóis/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Infecções por Escherichia coli/tratamento farmacológico , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Pirazóis/química , Pirazóis/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia
15.
Int J Biol Macromol ; 135: 725-733, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129210

RESUMO

Staphylopine is a newly identified broad-spectrum metallophore for metal acquisition, and it plays important roles in the survival and virulence of Staphylococcus aureus and other pathogens in the metal-scarce environment in hosts. CntK catalyzes the first step of staphylopine synthesis by converting L-histidine to D-histidine to provide an essential building block of staphylopine. Herein, the crystal structures of S. aureus CntK (SaCntK) and its C72S variant are determined at 1.82 and 1.58 Šresolution, respectively. SaCntK forms a homodimer and each subunit contains a two-domain α/ß structure. Its overall structure resembles diaminopimelate epimerase, although their sequence identities are lower than 22%. SaCntK is specific for histidine, whereas other proteinogenic amino acids, with the exception of arginine, do not show any binding with SaCntK. Structural modeling suggested that residues Asn16, Glu46, Gln47 and Glu208 are responsible for specific substrate binding, and their substitutions significantly reduced the binding of histidine to SaCntK. Structural modeling suggested SaCntK uses a two-base catalytic mechanism, which has been observed in many cofactor-independent racemases. Our study provides critical insights into the structure and functions of CntK in staphylopine synthesis, which makes it helpful for developing potential antibiotics targeting the staphylopine-mediated metal acquisition process in bacteria.


Assuntos
Histidina/metabolismo , Imidazóis/metabolismo , Metais/metabolismo , Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Staphylococcus aureus/enzimologia , Domínio Catalítico , Coenzimas/metabolismo , Cristalografia por Raios X , Evolução Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Staphylococcus aureus/metabolismo
16.
J Biol Chem ; 294(23): 9285-9294, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31018965

RESUMO

Oleate hydratases (OhyAs) belong to a large family of bacterial proteins catalyzing the hydration or isomerization of double bonds in unsaturated fatty acids. A Staphylococcus aureus gene (Sa0102) is predicted to encode an OhyA. Here, we recombinantly expressed and purified SaOhyA and found that it forms a homodimer that requires FAD for activity. SaOhyA hydrates only unsaturated fatty acids containing cis-9 double bonds, but not fatty acids with trans-9 double bonds or cis double bonds at other positions. SaOhyA products were not detected in S. aureus phospholipids and were released into the growth medium. S. aureus does not synthesize unsaturated fatty acids, and the SaOhyA substrates are derived from infection sites. Palmitoleate (16:1(9Z)) is a major mammalian skin-produced antimicrobial fatty acid that protects against S. aureus infection, and we observed that it is an SaOhyA substrate and that its hydroxylated derivative is not antimicrobial. Treatment of S. aureus with 24 µm 16:1(9Z) immediately arrested growth, followed by growth resumption after a lag period of 2 h. The ΔohyA mutant strain did not recover from the 16:1(9Z) challenge, and increasing SaOhyA expression using a plasmid system prevented the initial growth arrest. Challenging S. aureus with sapienic acid (16:1(6Z)), an antimicrobial fatty acid produced only by human skin, arrested growth without recovery in WT, ΔohyA, and SaOhyA-overexpressing strains. We conclude that SaOhyA protects S. aureus from palmitoleic acid, the antimicrobial unsaturated fatty acid produced by most mammals, and that sapienic acid, uniquely produced by humans, counters the OhyA-dependent bacterial defense mechanism.


Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Hidroliases/metabolismo , Staphylococcus aureus/enzimologia , Animais , Anti-Infecciosos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Graxos Insaturados/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidroliases/genética , Hidroliases/isolamento & purificação , Cinética , Pele/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Especificidade por Substrato
17.
PLoS Pathog ; 15(4): e1007723, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002736

RESUMO

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to ß-lactam antibiotics. The membrane-associated TagA glycosyltransferase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme's lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.


Assuntos
Proteínas de Bactérias/química , Parede Celular/metabolismo , Lipoproteínas/química , Staphylococcus aureus/enzimologia , Ácidos Teicoicos/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Lipoproteínas/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína
18.
Microbiology ; 165(5): 572-584, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30942689

RESUMO

The gold standard method for the creation of gene deletions in Staphylococcus aureus is homologous recombination using allelic exchange plasmids with a temperature-sensitive origin of replication. A knockout vector that contains regions of homology is first integrated into the chromosome of S. aureus by a single crossover event selected for at high temperatures (non-permissive for plasmid replication) and antibiotic selection. Next, the second crossover event is encouraged by growth without antibiotic selection at low temperature, leading at a certain frequency to the excision of the plasmid and the deletion of the gene of interest. To detect or encourage plasmid loss, either a beta-galactosidase screening method or, more typically, a counterselection step is used. We present here the adaptation of the counter-selectable marker pheS*, coding for a mutated subunit of the phenylalanine tRNA synthetase, for use in S. aureus. The PheS* protein variant allows for the incorporation of the toxic phenylalanine amino acid analogue para-chlorophenylalanine (PCPA) into proteins and the addition of 20-40 mM PCPA to rich media leads to drastic growth reduction for S. aureus and supplementing chemically defined medium with 2.5-5 mM PCPA leads to complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, we delete the magnesium transporter gene mgtE in S. aureus USA300 LAC* (SAUSA300_0910/SAUSA300_RS04895) and RN4220 (SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureus is mainly mediated by the presence of MgtE. This new plasmid will aid the efficient and easy creation of gene knockouts in S. aureus.


Assuntos
Proteínas de Bactérias/genética , Engenharia Genética , Fenilalanina-tRNA Ligase/genética , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Técnicas de Inativação de Genes , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Genoma Bacteriano , Humanos , Fenilalanina-tRNA Ligase/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo
19.
FEBS Open Bio ; 9(3): 420-427, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30868050

RESUMO

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil-DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil-DNA glycosylase. Interestingly, both uracil-DNA glycosylase (Staphylococcus aureus uracil-DNA glycosylase; SAUDG) and its inhibitor (S. aureus uracil-DNA glycosylase inhibitor; SAUGI) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil-DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI. Here, we investigated whether these mutations drastically perturb the interaction with SAUDG. To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these K d values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil-DNA glycosylase inhibitor proteins.


Assuntos
Substâncias Macromoleculares/análise , Mutação , Staphylococcus aureus/enzimologia , Uracila-DNA Glicosidase/química , Inibidores Enzimáticos/farmacologia , Substâncias Macromoleculares/antagonistas & inibidores , Substâncias Macromoleculares/metabolismo , Espectrometria de Massas , Modelos Moleculares , Uracila-DNA Glicosidase/antagonistas & inibidores , Uracila-DNA Glicosidase/metabolismo
20.
Eur J Med Chem ; 171: 209-220, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30925337

RESUMO

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis that is essential for bacterial survival. Therefore, FabH has been identified as an attractive target for the development of new antibacterial agents. We present here the discovery of a promising new series of Pyrazol-Benzimidazole amides with low toxicity and potent FabH inhibitory. Twenty-seven novel compounds have been synthesized, and all the compounds were characterized by 1H NMR, 13C NMR and MS. Afterwards they were evaluated for in-vitro antibacterial activities against E. coli, P. aeruginosa, B. subtilis and S. aureus, along with E. coli FabH inhibition and cytotoxicity test. Some compounds proved to be of low toxicity and potent, especially compound 31 exhibited the most potential to be a new drug with MIC of 0.49-0.98 µg/mL against the tested bacterial strains and IC50 of 1.22 µM against E. coli FabH. Eight analogues 16, 28, 30, 31, 33, 34, 35 and 36 with low range MIC against wild type Xanthomonas Campestris exhibited no inhibition against FabH-deficient mutant strain, which firmly proved the class of compounds arrived at antibacterial activity via interacting with FabH. In silico ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) evaluation also pointed out that these compounds are potential for druggability. Further, effective overall docking scores of all the compounds have been recorded, and docking simulation of compound 31 into E. coli FabH binding pocket has been conducted, where solid binding interactions has been identified.


Assuntos
Bacillus subtilis/enzimologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade
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