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1.
Methods Mol Biol ; 2341: 9-16, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264455

RESUMO

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.


Assuntos
N-Acetil-Muramil-L-Alanina Amidase/análise , Peptídeo Hidrolases/análise , Staphylococcus aureus/crescimento & desenvolvimento , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Hidrolases/metabolismo , Staphylococcus aureus/enzimologia
2.
Methods Mol Biol ; 2341: 17-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264456

RESUMO

Staphylococcal secreted nuclease contributes to S. aureus virulence by degrading neutrophil extracellular traps (NETs), which allows the bacterium to evade the host immune system and has also been shown to promote biofilm dispersal. In this chapter, two methods for detecting nuclease activity are described, both of which have increased sensitivity compared to the traditional nuclease agar method.


Assuntos
Proteínas de Bactérias/análise , Nuclease do Micrococo/análise , Salmão/genética , Staphylococcus aureus/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Armadilhas Extracelulares/metabolismo , Evasão da Resposta Imune , Masculino , Nuclease do Micrococo/metabolismo , Espermatozoides/química , Staphylococcus aureus/patogenicidade , Fatores de Virulência/análise , Fatores de Virulência/metabolismo
3.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074047

RESUMO

In this review, we chart the major milestones in the research progress on the DyP-type peroxidase family over the past decade. Though mainly distributed among bacteria and fungi, this family actually exhibits more widespread diversity. Advanced tertiary structural analyses have revealed common and different features among members of this family. Notably, the catalytic cycle for the peroxidase activity of DyP-type peroxidases appears to be different from that of other ubiquitous heme peroxidases. DyP-type peroxidases have also been reported to possess activities in addition to peroxidase function, including hydrolase or oxidase activity. They also show various cellular distributions, functioning not only inside cells but also outside of cells. Some are also cargo proteins of encapsulin. Unique, noteworthy functions include a key role in life-cycle switching in Streptomyces and the operation of an iron transport system in Staphylococcus aureus, Bacillus subtilis and Escherichia coli. We also present several probable physiological roles of DyP-type peroxidases that reflect the widespread distribution and function of these enzymes. Lignin degradation is the most common function attributed to DyP-type peroxidases, but their activity is not high compared with that of standard lignin-degrading enzymes. From an environmental standpoint, degradation of natural antifungal anthraquinone compounds is a specific focus of DyP-type peroxidase research. Considered in its totality, the DyP-type peroxidase family offers a rich source of diverse and attractive materials for research scientists.


Assuntos
Antraquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Lignina/metabolismo , Peroxidases/química , Peroxidases/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Catálise , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Fungos/crescimento & desenvolvimento , Ferro/metabolismo , Oxirredução , Filogenia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismo
4.
Science ; 372(6547): 1169-1175, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112687

RESUMO

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.


Assuntos
Antibacterianos/farmacologia , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Biofilmes , Cristalografia por Raios X , Cistationina gama-Liase/química , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Descoberta de Drogas , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Tolerância a Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento
5.
DNA Repair (Amst) ; 105: 103160, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34192601

RESUMO

GO system is part of base excision DNA repair and is required for the correct repair of 8-oxoguanine (8-oxoG), one of the most abundant oxidative lesions. Due to the ability of 8-oxoG to mispair with A, this base is highly mutagenic, and its repair requires two enzymes: Fpg that removes 8-oxoG from 8-oxoG:C pairs, and MutY that excises the normal A from 8-oxoG:A mispairs. Here we characterize the properties of putative GO system DNA glycosylases from Staphylococcus aureus, an important human opportunistic pathogen that causes hospital infections and presents a serious health concern due to quick spread of antibiotic-resistant strains. In addition to Fpg and MutY from the reference NCTC 8325 strain (SauFpg1 and SauMutY), we have also studied an Fpg homolog from a multidrug-resistant C0673 isolate (SauFpg2), which is different from SauFpg1 in its sequence. Both SauFpg enzymes showed the highest activity at pH 7.0-9.0 and NaCl concentrations 25-75 mM (SauFpg1) or 50-100 mM (SauFpg2), whereas SauMutY was active at a broad pH range and had a salt optimum at ∼75 mM NaCl. Both SauFpg1 and SauFpg2 bound and cleaved duplexes containing 8-oxoG, 5-hydroxyuracil, 5,6-dihydrouracil or apurinic/apyrimidinic site paired with C, T, or G, but not with A. For SauFpg1 and SauFpg2, 8-oxoG was the best substrate tested, and 5,6-dihydrouracil was the worst one. SauMutY efficiently excised adenine from duplex substrates containing A:8-oxoG or A:G pairs. SauFpg enzymes were readily trapped on DNA by NaBH4 treatment, indicating formation of a Schiff base reaction intermediate. Surprisingly, SauMutY was also trapped significantly better than its E. coli homolog. All three S. aureus GO glycosylases drastically reduced spontaneous mutagenesis when expressed in an fpg mutY E. coli double mutant. Overall, we conclude that S. aureus possesses an active GO system, which could possibly be targeted for sensitization of this pathogen to oxidative stress.


Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Guanina/análogos & derivados , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Glicosilases/genética , DNA Bacteriano/metabolismo , DNA-Formamidopirimidina Glicosilase/genética , Guanina/metabolismo , Concentração de Íons de Hidrogênio , Filogenia , Alinhamento de Sequência , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Especificidade por Substrato
6.
Nucleic Acids Res ; 49(11): 6587-6595, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107040

RESUMO

Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9-sgRNA-dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.


Assuntos
Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Proteína 9 Associada à CRISPR/química , Staphylococcus aureus/enzimologia , Regulação Alostérica , Cristalografia por Raios X , DNA/química , Modelos Moleculares , Domínios Proteicos , RNA/química
7.
Infect Immun ; 89(8): e0014621, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34001560

RESUMO

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Metionina Sulfóxido Redutases/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/imunologia , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Peróxido de Hidrogênio/metabolismo , Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/imunologia , Camundongos , Viabilidade Microbiana/imunologia , Mutação , Oxirredução , Estresse Oxidativo , Staphylococcus aureus/genética
8.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918623

RESUMO

Compounds targeting bacterial topoisomerases are of interest for the development of antibacterial agents. Our previous studies culminated in the synthesis and characterization of small-molecular weight thiosemicarbazides as the initial prototypes of a novel class of gyrase and topoisomerase IV inhibitors. To expand these findings with further details on the mode of action of the most potent compounds, enzymatic studies combined with a molecular docking approach were carried out, the results of which are presented herein. The biochemical assay for 1-(indol-2-oyl)-4-(4-nitrophenyl) thiosemicarbazide (4) and 4-benzoyl-1-(indol-2-oyl) thiosemicarbazide (7), showing strong inhibitory activity against Staphylococcus aureus topoisomerase IV, confirmed that these compounds reduce the ability of the ParE subunit to hydrolyze ATP rather than act by stabilizing the cleavage complex. Compound 7 showed better antibacterial activity than compound 4 against clinical strains of S. aureus and representatives of the Mycobacterium genus. In vivo studies using time-lapse microfluidic microscopy, which allowed for the monitoring of fluorescently labelled replisomes, revealed that compound 7 caused an extension of the replication process duration in Mycobacterium smegmatis, as well as the growth arrest of bacterial cells. Despite some similarities to the mechanism of action of novobiocin, these compounds show additional, unique properties, and can thus be considered a novel group of inhibitors of the ATPase activity of bacterial type IIA topoisomerases.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Semicarbazidas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Antibacterianos/química , Sítios de Ligação , DNA Girase/química , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Semicarbazidas/química , Relação Estrutura-Atividade , Inibidores da Topoisomerase/química , Inibidores da Topoisomerase/farmacologia
9.
J Microbiol ; 59(6): 584-589, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33877576

RESUMO

The SbcCD complex is an essential component of the DNA double-strand break (DSB) repair system in bacteria. The bacterial SbcCD complex recognizes and cleaves the DNA ends in DSBs by ATP-dependent endo- and exonuclease activities as an early step of the DNA repair process. SbcD consists of nuclease, capping, and helix-loop-helix domains. Here, we present the crystal structure of a SbcD fragment from Staphylococcus aureus, which contained nuclease and capping domains, at a resolution of 2.9 Å. This structure shows a dimeric assembly similar to that of the corresponding domains of SbcD from Escherichia coli. The S. aureus SbcD fragment exhibited endonuclease activities on supercoiled DNA and exonuclease activity on linear and nicked DNA. This study contributes to the understanding of the molecular basis for how bacteria can resist sterilizing treatment, causing DNA damage.


Assuntos
Proteínas de Bactérias/química , Desoxirribonucleases/química , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalização , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Bacteriano/genética , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Conformação Proteica , Domínios Proteicos , Staphylococcus aureus/química , Staphylococcus aureus/genética
10.
Front Immunol ; 12: 651060, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33833764

RESUMO

In cystic fibrosis (CF) infectious and allergic airway inflammation cause pulmonary exacerbations that destroy the lungs. Staphylococcus aureus is a common long-term colonizer and cause of recurrent airway infections in CF. The pathogen is also associated with respiratory allergy; especially the staphylococcal serine protease-like proteins (Spls) can induce type 2 immune responses in humans and mice. We measured the serum IgE levels specific to 7 proteases of S. aureus by ELISA, targeting 5 Spls (76 CF patients and 46 controls) and the staphopains A and B (16 CF patients and 46 controls). Then we compared cytokine release and phenotype of T cells that had been stimulated with Spls between 5 CF patients and 5 controls. CF patients had strongly increased serum IgE binding to all Spls but not to the staphopains. Compared to healthy controls, their Spl-stimulated T cells released more type 2 cytokines (IL-4, IL-5, IL-13) and more IL-6 with no difference in the secretion of type 1- or type 3 cytokines (IFNγ, IL-17A, IL-17F). IL-10 production was low in CF T cells. The phenotype of the Spl-exposed T cells shifted towards a Th2 or Th17 profile in CF but to a Th1 profile in controls. Sensitization to S. aureus Spls is common in CF. This discovery could explain episodes of allergic inflammation of hitherto unknown causation in CF and extend the diagnostic and therapeutic portfolio.


Assuntos
Proteínas de Bactérias/imunologia , Fibrose Cística/imunologia , Hipersensibilidade/microbiologia , Serina Proteases/imunologia , Infecções Estafilocócicas/imunologia , Adolescente , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Fibrose Cística/sangue , Fibrose Cística/microbiologia , Feminino , Voluntários Saudáveis , Interações Hospedeiro-Patógeno/imunologia , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Cultura Primária de Células , Serina Proteases/metabolismo , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/imunologia , Linfócitos T/imunologia , Adulto Jovem
11.
FASEB J ; 35(5): e21575, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33826776

RESUMO

Staphylopine (StP) and other nicotianamine-like metallophores are crucial for many pathogens to acquire the transition metals from hosts during invasion. CntL from Staphylococcus aureus (SaCntL) catalyzes the condensation of the 2-aminobutyrate (Ab) moiety of S-adenosylmethionine (SAM) with D-histidine in the biosynthesis of StP. Here, we report the crystal structures of SaCntL in complex with either SAM or two products. The structure of SaCntL consists of an N-terminal four-helix bundle (holding catalytic residue E84) and a C-terminal Rossmann fold (binding the substrates). The sequence connecting the N- and C-terminal domains (N-C linker) in SaCntL was found to undergo conformational alternation between open and closed states. Our structural and biochemical analyses suggested that this intrinsically dynamic interdomain linker forms an additional structural module that plays essential roles in ligand diffusion, recognition, and catalysis. We confirmed that SaCntL stereoselectively carries out the catalysis of D-His but not its enantiomer, L-His, and we found that the N-C linker and active site of SaCntL could accommodate both enantiomers. SaCntL is likely able to bind L-His without catalysis, and as a result, L-His could show inhibitory effects toward SaCntL. These findings provide critical structural and mechanistic insights into CntL, which facilitates a better understanding of the biosynthesis of nicotianamine-like metallophores and the discovery of inhibitors of this process.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Imidazóis/metabolismo , Staphylococcus aureus/enzimologia , Transferases/química , Transferases/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Conformação Proteica
12.
ACS Infect Dis ; 7(4): 746-758, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33710875

RESUMO

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms. Here, we investigated the effect of a 4'-pyridone substituent in the context of the slow tight-binding inhibitor SKTS1 on the inhibition of the Staphylococcus aureus enoyl-ACP-reductase saFabI and the closely related isoenzyme from Mycobacterium tuberculosis, InhA, and explored a new interaction site of DPE inhibitors within the substrate-binding pocket. Using high-resolution crystal structures of both complexes in combination with molecular dynamics (MD) simulations, kinetic measurements, and quantum mechanical (QM) calculations, we provide evidence that the 4'-pyridone substituent adopts different tautomeric forms when bound to the two ENRs. We furthermore elucidate the structural determinants leading to significant differences in the residence time of SKTS1 on both enzymes.


Assuntos
Inibidores Enzimáticos/farmacologia , Isoenzimas , Oxirredutases/antagonistas & inibidores , Isomerismo , Mycobacterium tuberculosis/enzimologia , Staphylococcus aureus/enzimologia
13.
Vet Res ; 52(1): 41, 2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33676576

RESUMO

MicroRNAs (miRNAs) are short, non-coding RNAs, 21-23 nucleotides in length which are known to regulate biological processes that greatly impact immune system activity. The aim of the study was to compare the miRNA expression in non-infected (H) mammary gland parenchyma samples with that of glands infected with coagulase-positive staphylococci (CoPS) or coagulase-negative staphylococci (CoNS) using next-generation sequencing. The miRNA profile of the parenchyma was found to change during mastitis, with its profile depending on the type of pathogen. Comparing the CoPS and H groups, 256 known and 260 potentially new miRNAs were identified, including 32 that were differentially expressed (p ≤ 0.05), of which 27 were upregulated and 5 downregulated. Comparing the CoNS and H groups, 242 known and 171 new unique miRNAs were identified: 10 were upregulated (p ≤ 0.05), and 2 downregulated (p ≤ 0.05). In addition, comparing CoPS with H and CoNS with H, 5 Kyoto Encyclopedia of Genes and Genomes pathways were identified; in both comparisons, differentially-expressed miRNAs were associated with the bacterial invasion of epithelial cells and focal adhesion pathways. Four gene ontology terms were identified in each comparison, with 2 being common to both immune system processes and signal transduction. Our results indicate that miRNAs, especially miR-99 and miR-182, play an essential role in the epigenetic regulation of a range of cellular processes, including immunological systems bacterial growth in dendritic cells and disease pathogenesis (miR-99), DNA repair and tumor progression (miR-182).


Assuntos
Glândulas Mamárias Animais/metabolismo , Mastite Bovina/microbiologia , MicroRNAs/genética , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia , Transcriptoma , Animais , Bovinos , Coagulase/metabolismo , Escherichia coli/fisiologia , Infecções por Escherichia coli/microbiologia , Feminino , Perfilação da Expressão Gênica/veterinária , Células do Mesofilo/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia
14.
Bioorg Med Chem Lett ; 40: 127966, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33744441

RESUMO

Antibiotic resistance represents one of the biggest public health challenges in the last few years. Mur ligases (MurC-MurF) are involved in the synthesis of UDP-N-acetylmuramyl-pentapeptide, the main building block of bacterial peptidoglycan polymer. They are essential for the survival of bacteria and therefore important antibacterial targets. We report herein the synthesis and structure-activity relationships of Mur ligases inhibitors with an azastilbene scaffold. Several compounds showed promising inhibitory potencies against multiple ligases and one compound also possessed moderate antibacterial activity. These results represent a solid ground for further development and optimization of structurally novel antimicrobial agents to combat the rising bacterial resistance.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Benzilideno/farmacologia , Inibidores Enzimáticos/farmacologia , Peptídeo Sintases/antagonistas & inibidores , Piridinas/farmacologia , Antibacterianos/síntese química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeo Sintases/metabolismo , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade
15.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672341

RESUMO

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.


Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Metionina/metabolismo , Modelos Moleculares , Peptídeo Hidrolases/genética , Peptídeos/química , Peptídeos/metabolismo , Especificidade por Substrato
16.
mBio ; 12(1)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622717

RESUMO

Staphylococcus aureus controls the progression of infection through the coordinated production of extracellular proteases, which selectively modulate virulence determinant stability. This is evidenced by our previous finding that a protease-null strain has a hypervirulent phenotype in a murine model of sepsis, resulting from the unchecked accumulation of virulence factors. Here, we dissect the individual roles of these proteases by constructing and assessing the pathogenic potential of a combinatorial protease mutant library. When strains were constructed bearing increasing numbers of secreted proteases, we observed a variable impact on infectious capacity, where some exhibited hypervirulence, while others phenocopied the wild-type. The common thread for hypervirulent strains was that each lacked both aureolysin and staphopain A. Upon assessment, we found that the combined loss of these two enzymes alone was necessary and sufficient to engender hypervirulence. Using proteomics, we identified a number of important secreted factors, including SPIN, LukA, Sbi, SEK, and PSMα4, as well as an uncharacterized chitinase-related protein (SAUSA300_0964), to be overrepresented in both the aur scpA and the protease-null mutants. When assessing the virulence of aur scpA SAUSA300_0964 and aur scpA lukA mutants, we found that hypervirulence was completely eliminated, whereas aur scpA spn and aur scpA sek strains elicited aggressive infections akin to the protease double mutant. Collectively, our findings shed light on the influence of extracellular proteases in controlling the infectious process and identifies SAUSA300_0964 as an important new component of the S. aureus virulence factor arsenal.IMPORTANCE A key feature of the pathogenic success of S. aureus is the myriad virulence factors encoded within its genome. These are subject to multifactorial control, ensuring their timely production only within an intended infectious niche. A key node in this network of control is the secreted proteases of S. aureus, who specifically and selectively modulate virulence factor stability. In our previous work we demonstrated that deletion of all 10 secreted proteases results in hypervirulence, since virulence factors exist unchecked, leading to overly aggressive infections. Here, using a combinatorial collection of protease mutants, we reveal that deletion of aureolysin and staphopain A is necessary and sufficient to elicit hypervirulence. Using proteomic techniques, we identify the collection of virulence factors that accumulate in hypervirulent protease mutants, and demonstrate that a well-known toxin (LukA) and an entirely novel secreted element (SAUSA300_0964) are the leading contributors to deadly infections observed in protease-lacking strains.


Assuntos
Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteômica/métodos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismo , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Virulência , Fatores de Virulência/genética
17.
Biochem Biophys Res Commun ; 545: 98-104, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33548630

RESUMO

A large class of bacterial RNA polymerase (RNAP) from low-G + C-content Gram-positive bacterial strains, such as the major human pathogen Staphylococcus aureus, not only contain five conserved subunits (αI, αII, ß, ß' and ω), but also has a δ subunit. Despite being first identified as unique, Gram-positive specific component of RNAP apoenzyme more than 30 years ago and reported to be essential for transcription, the structural basis and molecular mechanism of δ subunit in the regulation of transcription remain poorly understood. Here, we performed structural analyses, site-directed mutagenesis and biochemical assays to uncover the interactions of S. aureus δ subunit with RNAP core enzyme and DNA towards the understanding of its role in transcription regulation. Microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA) of the wild-type and mutated S. aureus δ subunit revealed the N-terminal domain of δ subunit directly binds to the ß' jaw of S. aureus RNAP (SauRNAP), identified the key amino acid residues (F58, D61, D65, R67 and W81) of δ subunit involving in the binding with SauRNAP core enzyme, and uncovered the δ subunit C-terminal domain interferes with the interaction between DNA and SauRNAP core enzyme, by which transcription is regulated. Our results provide an excellent starting point for understanding the unique regulatory role and physiological function of δ subunit on transcription regulation in Gram-positive bacteria.


Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Bacterianos , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/genética , Transcrição Genética
18.
Virulence ; 12(1): 584-600, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33538230

RESUMO

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.


Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/genética , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Animais , Proteínas de Bactérias/biossíntese , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Mutação , Fenótipo , Sepse/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo , Virulência
19.
Bioorg Chem ; 109: 104704, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33609915

RESUMO

New substituted pyrazolone and dipyrazolotriazine derivatives have been synthesized, designed and well characterized as promising dual antimicrobial/antioxidant agents to overcome multidrug resistant bacteria (MDR), oxidative stress and their related diseases. Among all strains, S. aureus was found to be the most susceptible for all compounds except 10b and 12b. Out of the three investigated series, sulfonamide analogues 5a-c displayed excellent antibacterial activity with 5b (MIC = 7.61 µM) and 5a (MIC = 8.98 µM) displaying activity that exceeds the reference drug tetracycline (MIC = 11.77 µM). The same sulfonamide derivatives 5a-c demonstrates high ABTS scavenging capacity comparable to standard. Moreover, the structure-activity relationship (SAR) revealed that benzenesulfonamide is a crucial group for enhancing activity. Molecular docking studies of the potent analogues were performed by targeting the crystal structures of S. aureus tyrosyl-tRNA synthetase and human peroxiredoxin-5 enzymes and the obtained results supported well the in vitro data revealing stronger binding interactions. Pharmacokinetics prediction together with modeling outcomes suggests that our sulfonamide derivatives may serve as useful lead compounds for the treatment of infectious disease.


Assuntos
Simulação de Acoplamento Molecular , Peroxirredoxinas/antagonistas & inibidores , Pirazolonas/química , Pirazolonas/farmacologia , Triazinas/farmacologia , Tirosina-tRNA Ligase/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade , Triazinas/química
20.
Eur J Med Chem ; 213: 113200, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524686

RESUMO

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.


Assuntos
Trifosfato de Adenosina/farmacologia , Antibacterianos/farmacologia , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Antibacterianos/síntese química , Antibacterianos/química , Cristalografia por Raios X , DNA Topoisomerase IV/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Relação Estrutura-Atividade
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