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1.
J Enzyme Inhib Med Chem ; 35(1): 584-597, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31992093

RESUMO

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of "selectivity index" in comparison to 1.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Quinolinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Quinolinas/síntese química , Quinolinas/química , Staphylococcus aureus/metabolismo , Relação Estrutura-Atividade
2.
Pol J Microbiol ; 68(4): 477-491, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880892

RESUMO

This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 - 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 ­ 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Farmacorresistência Bacteriana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , beta-Lactamas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriólise/efeitos dos fármacos , Cloxacilina/farmacologia , Quimioterapia Combinada , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Miconazol/farmacologia , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/citologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Tioridazina/farmacologia
3.
Pol J Microbiol ; 68(4): 541-548, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880897

RESUMO

Analysis of the epidemiology of Staphylococcus aureus (SA) ocular infections and virulence factors of the isolates with a special emphasis on their drug resistance, and the ability of biofilm formation. In a period from 2009 to 2013, 83 isolates of SA were prospectively collected and preserved in a multicenter laboratory-based study carried out in southern Poland. Epidemiological, phenotypic, and genotypic analyses were performed. The resistance and virulence genes were analyzed. Screening for the biofilm formation was provided. Among the materials derived from ocular infections from 456 patients, SA was found in 18.2% (n = 83) of cases (one SA isolate per one patient). Most infections were identified in the age group of over 65 years (OR 8.4 95%CI; 1.03-68.49). The majority of patients (73.4%) were hospitalized. Among the virulence and resistance genes, the most frequently detected were the lukE (72.2%, n = 60) and ermA (15.6%, n = 13) genes. A positive result of the CRA test (the ability of biofilm formation) was found in 66.2% (n = 55) of isolates. Among the strains under study, 6.0% (n = 5) had the methicillin-resistant Staphylococcus aureus phenotype, and 26.5% (n = 22) had the macrolide-lincosamide-streptogramin B phenotype. In 48 (57.8%) isolates the neomycin resistance was revealed. All isolates under study were sensitive to vancomycin. The population most susceptible to ocular SA infections consists of hospitalized patients aged 65 and more. The SA strains under study showed the increased ability to biofilm formation. In the strains tested, high susceptibility to chloramphenicol and fluoroquinolones was demonstrated. However, the high level of drug resistance to neomycin detected in this study among SA isolates and the blood-ocular barrier makes it difficult to treat ocular infections.Analysis of the epidemiology of Staphylococcus aureus (SA) ocular infections and virulence factors of the isolates with a special emphasis on their drug resistance, and the ability of biofilm formation. In a period from 2009 to 2013, 83 isolates of SA were prospectively collected and preserved in a multicenter laboratory-based study carried out in southern Poland. Epidemiological, phenotypic, and genotypic analyses were performed. The resistance and virulence genes were analyzed. Screening for the biofilm formation was provided. Among the materials derived from ocular infections from 456 patients, SA was found in 18.2% (n = 83) of cases (one SA isolate per one patient). Most infections were identified in the age group of over 65 years (OR 8.4 95%CI; 1.03-68.49). The majority of patients (73.4%) were hospitalized. Among the virulence and resistance genes, the most frequently detected were the lukE (72.2%, n = 60) and ermA (15.6%, n = 13) genes. A positive result of the CRA test (the ability of biofilm formation) was found in 66.2% (n = 55) of isolates. Among the strains under study, 6.0% (n = 5) had the methicillin-resistant Staphylococcus aureus phenotype, and 26.5% (n = 22) had the macrolide-lincosamide-streptogramin B phenotype. In 48 (57.8%) isolates the neomycin resistance was revealed. All isolates under study were sensitive to vancomycin. The population most susceptible to ocular SA infections consists of hospitalized patients aged 65 and more. The SA strains under study showed the increased ability to biofilm formation. In the strains tested, high susceptibility to chloramphenicol and fluoroquinolones was demonstrated. However, the high level of drug resistance to neomycin detected in this study among SA isolates and the blood-ocular barrier makes it difficult to treat ocular infections.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Infecções Oculares/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Fatores de Virulência/genética , Adolescente , Adulto , Idoso , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Criança , Infecções Oculares/epidemiologia , Feminino , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Polônia/epidemiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo , Adulto Jovem
4.
Int. microbiol ; 22(4): 411-417, dic. 2019. graf, tab
Artigo em Inglês | IBECS | ID: ibc-185059

RESUMO

Glycopeptides, particularly the cell wall-acting antibiotic vancomycin, are the safest cure for methicillin-resistant Staphylococcus aureus. The aim of this study was to evaluate nonsusceptibility of clinical isolates of S. aureus to vancomycin and investigate mutations in vraSR, a cell wall synthesis regulator gene, in vancomycin-resistant strains. Susceptibility of 110 clinical strains of S. aureus to methicillin and vancomycin were determined using disc diffusion method and determination of minimum inhibitory concentration, respectively. Presence of mecA and vanA genes was determined by PCR. Determination of spa types and mutations of the vraSR gene in vancomycin nonsusceptible isolates were assessed by PCR-sequencing analyses. In total, 47 isolates (42.73%) were recognized as MRSA, three (2.73%) strains were resistant to vancomycin, and eight (7.27%) strains were vancomycin intermediates. The MIC of vancomycin was 4-64 μg/ml in these isolates. All vancomycin nonsusceptible S. aureus strains were mecA positive and one isolate was positive for the vanA gene. Spa type t030 was found as the most common type. In vraSR sequence analysis, all 11 vancomycin nonsusceptible isolates had the D59E mutation in the vraR and E45G in vraS genes. R117H, R121S, and R121I are the other identified missense mutations in the vraR gene. The identification of a high percentage of MRSA and presence of VRSA and VISA isolates is a serious warning about the treatment of future MRSA infections and reveals the need for new and effective therapeutic agents


No disponible


Assuntos
Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/isolamento & purificação , Staphylococcus aureus/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Vancomicina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Irã (Geográfico) , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/tratamento farmacológico
5.
Infect Immun ; 88(1)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31685545

RESUMO

Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN.


Assuntos
Imunidade Adaptativa , Células Dendríticas/imunologia , Peptidoglicano/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia , Subpopulações de Linfócitos T/imunologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Fatores Imunológicos/metabolismo , Staphylococcus aureus/metabolismo
6.
J Agric Food Chem ; 67(48): 13195-13211, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31702908

RESUMO

The increase in the incidence of antibiotic-resistant Staphylococcus aureus (S. aureus) associated infections necessitates the urgent development of novel therapeutic strategies and antibacterial drugs. Antivirulence strategy is an especially compelling alternative strategy due to its low selective pressure for the development of drug resistance in bacteria. Plants and microorganisms are not only important food and medicinal resources but also serve as sources for the discovery of natural products that target bacterial virulence factors. This review discusses the mechanisms of the major virulence factors of S. aureus, including the accessory gene regulator quorum-sensing system, bacterial biofilm formation, α-hemolysin, sortase A, and staphyloxanthin. We also provide an overview of natural products isolated from plants and microorganisms with activity against the major virulence factors of S. aureus and their adjuvant effects on existing antibiotics to overcome antibiotic-resistant S. aureus. Finally, the limitations and solutions of these antivirulence compounds are discussed, which will help in the development of novel antibacterial drugs against antibiotic-resistant S. aureus.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Produtos Biológicos/farmacologia , Farmacorresistência Bacteriana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Fatores de Virulência/antagonistas & inibidores , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
8.
Medicine (Baltimore) ; 98(38): e17185, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31567961

RESUMO

Infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus (PVL-SA) mostly present as recurrent skin abscesses and furunculosis. However, life-threatening infections (eg, necrotizing pneumonia, necrotizing fasciitis, and osteomyelitis) caused by PVL-SA have also been reported.We assessed the clinical phenotype, frequency, clinical implications (surgery, length of treatment in hospitals/intensive care units, and antibiotic treatments), and potential preventability of severe PVL-SA infections in children.Total, 75 children treated for PVL-SA infections in our in- and outpatient units from 2012 to 2017 were included in this retrospective study.Ten out of 75 children contracted severe infections (PVL-methicillin resistant S aureus n = 4) including necrotizing pneumonia (n = 4), necrotizing fasciitis (n = 2), pyomyositis (n = 2; including 1 patient who also had pneumonia), mastoiditis with cerebellitis (n = 1), preorbital cellulitis (n = 1), and recurrent deep furunculosis in an immunosuppressed patient (n = 1). Specific complications of PVL-SA infections were venous thrombosis (n = 2), sepsis (n = 5), respiratory failure (n = 5), and acute respiratory distress syndrome (n = 3). The median duration of hospital stay was 14 days (range 5-52 days). In 6 out of 10 patients a history suggestive for PVL-SA colonization in the patient or close family members before hospital admission was identified.PVL-SA causes severe to life-threatening infections requiring lengthy treatments in hospital in a substantial percentage of symptomatic PVL-SA colonized children. More than 50% of severe infections might be prevented by prompt testing for PVL-SA in individuals with a history of abscesses or furunculosis, followed by decolonization measures.


Assuntos
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Leucocidinas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Tempo de Internação , Masculino , Pneumonia Necrosante/microbiologia , Estudos Retrospectivos , Infecções dos Tecidos Moles/microbiologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/patologia , Infecções Estafilocócicas/terapia
9.
J Food Sci ; 84(11): 3233-3240, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31618461

RESUMO

Food poisoning caused by Staphylococcus aureus is responsible for staphylococcal enterotoxin (SE) produced in foods. Staphylococcal food poisoning is mostly caused by staphylococcal enterotoxin type A (SEA) among SEs. Growth/no growth for S. aureus under various environmental conditions was well studied with a logistic regression model so far. Recently we successfully described the boundaries of SEA production and growth of S. aureus in broth at various temperatures and salt concentrations with the model. In this study, the effects of lactic acid and salt on SEA production and growth of S. aureus was quantitatively studied. Consequently the boundaries of SEA production and growth of S. aureus cocktail in broth at various combinations of salt concentrations and pH values that were adjusted with lactic acid were successfully described with a logistic regression model. Here the cocktail was incubated in stationary culture at 30 °C and 10 °C. The maximum toxin production and cell growth of the cocktail were observed both at 5% salt in the pH range from 4.5 to 7.0. Also, the characteristics of individual strains of the cocktail in SEA production and growth at 30 °C and 10 °C were found to be specific to the strains. The present study revealed the effect of lactic acid and salt on SEA production and growth of S. aureus as well as the variety of SEA production and growth of S. aureus strains. These results would become useful information in food industry to prevent staphylococcal food poisoning. PRACTICAL APPLICATION: Boundaries of enterotoxin A production/no production and growth/no growth of staphylococcal cocktail at various combinations of pHs adjusted with lactic acid and salt concentrations were well described with a logistic regression model. The maximum toxin production and cell growth were observed both at 5% salt in the pH range from 4.5 to 7.0. A variety of the toxin production and cell growth were observed in terms of pH and salt concentration among individual strains of the cocktail.


Assuntos
Enterotoxinas/biossíntese , Ácido Láctico/farmacologia , Cloreto de Sódio/farmacologia , Staphylococcus aureus/crescimento & desenvolvimento , Microbiologia de Alimentos , Intoxicação Alimentar Estafilocócica/etiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Temperatura Ambiente
10.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527127

RESUMO

Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium. Using a fluorescence resonance energy transfer (FRET)-based assay, we detected thermonuclease activity of S. aureus biofilms grown in Iscove's modified Dulbecco's medium (IMDM) earlier than that of widely studied biofilms grown in tryptic soy broth (TSB). The thermonuclease found was Nuc1, confirmed by mass spectrometry and competitive Luminex assay. These results indicate that biofilm development in IMDM may not rely on eDNA for structural stability. A bacterial viability assay in combination with wheat germ agglutinin (WGA) staining confirmed the accumulation of dead cells and eDNA in biofilms grown in TSB. However, in biofilms grown in IMDM, minimal amounts of eDNA were found; instead, polysaccharide intercellular adhesin (PIA) was detected. To investigate if this early production of thermonuclease plays a role in immune modulation by biofilm, we studied the effect of thermonuclease on human neutrophil extracellular trap (NET) formation using a nuc knockout and complemented strain. We confirmed that thermonuclease produced by early-stage biofilms grown in IMDM degraded biofilm-induced NETs. Additionally, neither the presence of biofilms nor thermonuclease stimulated an increase in reactive oxygen species (ROS) production by neutrophils. Our findings indicated that S. aureus, during the early stages of biofilm formation, actively evades the host immune responses by producing thermonuclease.


Assuntos
Biofilmes/crescimento & desenvolvimento , Armadilhas Extracelulares/metabolismo , Nuclease do Micrococo/metabolismo , Neutrófilos/imunologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Transferência Ressonante de Energia de Fluorescência , Humanos , Viabilidade Microbiana , Polissacarídeos Bacterianos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/metabolismo
11.
PLoS Genet ; 15(8): e1008336, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31415562

RESUMO

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Regulon/genética , Staphylococcus aureus/metabolismo , Proteínas de Transporte de Cátions/genética , Mutação com Ganho de Função , Homeostase , Staphylococcus aureus/genética
12.
Int J Mol Sci ; 20(16)2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430864

RESUMO

The use and misuse of antibiotics has resulted in critical conditions for drug-resistant bacteria emergency, accelerating the development of antimicrobial resistance (AMR). In this context, the co-administration of an antibiotic with a compound able to restore sufficient antibacterial activity may be a successful strategy. In particular, the identification of efflux pump inhibitors (EPIs) holds promise for new antibiotic resistance breakers (ARBs). Indeed, bacterial efflux pumps have a key role in AMR development; for instance, NorA efflux pump contributes to Staphylococcus aureus (S. aureus) resistance against fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from the cells. Even though NorA efflux pump is known to be a potential target for EPIs development, the absence of structural information about this protein and the little knowledge available on its mechanism of action have strongly hampered rational drug discovery efforts in this area. In the present work, we investigated at the molecular level the substrate recognition pathway of NorA through a Supervised Molecular Dynamics (SuMD) approach, using a NorA homology model. Specific amino acids were identified as playing a key role in the efflux pump-mediated extrusion of its substrate, paving the way for a deeper understanding of both the mechanisms of action and the inhibition of such efflux pumps.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Ciprofloxacino/farmacologia , Farmacorresistência Bacteriana Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo
13.
Int J Food Microbiol ; 308: 108291, 2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31437692

RESUMO

Biocontrol of Staphylococcus aureus by lactic acid bacteria can be considered as a feasible alternative to the use of chemicals in foods, but the mechanisms underlying this antagonistic interaction remains poorly understood. This study aimed to evaluate the impact of a LAB species (Enterococcus faecalis) over the growth, enterotoxin production and gene expression of S. aureus under experimental conditions. E. faecalis 41FL1 and S. aureus ATCC 29213 were inoculated isolated and together in brain heart infusion (BHI) at 30 °C and in a fresh cheese model at 15 °C: microbial populations were monitored by culture plating, production of classical staphylococcal enterotoxins (SEs) was verified by an ELISA assay, expression of S. aureus genes (virulence, transcriptional regulation and central carbon metabolism) was investigated by quantitative real-time PCR, and pH and contents of water-soluble metabolites in both matrices were measured. S. aureus growth was inhibited in co-cultures assays, with a 2.02-log reduction in BHI and a 3.39-log reduction in cheese model compared to respective single cultures. Classical SEs were detected in S. aureus single culture assays (BHI and cheese), in BHI inoculated with both strains after 48 h of incubation, but not detected in co-inoculated cheeses. pH in all matrices containing E. faecalis reached lower values than in matrices containing S. aureus alone, due to lactate production by E. faecalis. Expression of genes coding for transcription regulators (ccpA and rex) and enzymes involved in central carbon metabolism (alsD and citZ) was mostly upregulated in co-inoculated cheeses, whereas expression of several virulence determinants (agrC, hld, hla, entA and spa) was strongly downregulated. This study provides relevant data on the behaviour of S. aureus in the presence of competing microbiota and support the use of controlled population dominance by LAB as an effective biopreservation strategy to ensuring food safety.


Assuntos
Antibiose/fisiologia , Agentes de Controle Biológico/metabolismo , Queijo/microbiologia , Enterococcus faecalis/metabolismo , Enterotoxinas/biossíntese , Staphylococcus aureus/metabolismo , Animais , Técnicas de Cocultura , Enterotoxinas/genética , Inocuidade dos Alimentos/métodos , Expressão Gênica , Leite/microbiologia , Infecções Estafilocócicas/prevenção & controle , Fatores de Virulência/metabolismo
14.
Int J Med Microbiol ; 309(6): 151333, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31362856

RESUMO

Staphylococcus aureus produces different secondary cell wall glycopolymers such as wall teichoic acids (WTA) and capsular polysaccharides (CP). These structures play an important role in S. aureus colonization, pathogenesis and bacterial evasion of the host immune defences. To fulfil their diverse functions, biosynthesis of both glycopolymers has to be tightly controlled. Regulation of WTA biosynthesis and modification is only partially understood. The transcription factor MgrA and the two-component systems (TCS) Agr, GraRS, and ArlRS control WTA export, chain-length and modification. CP synthesis is determined by transcriptional and post-transcriptional regulatory circuits. On the transcriptional level expression of the capA-P operon is mainly driven by the alternative Sigma factor B and modulated by several transcriptional factors and TCS. Post-transcriptional mechanisms are in place to avoid conflict between precursor usage by the CP synthesis machinery and the synthesis machinery of other cell wall glycopolymers. The complex interplay of these regulatory systems determines the peculiar, strictly temporal expression of CP in the late growth phase and the high degree of phenotypic heterogeneity. Differential expression of CP, WTA and its modification systems during infection and colonisation are likely important for disease development, immune escape and survival within the host.


Assuntos
Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/genética , Ácidos Teicoicos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade
15.
J Chromatogr A ; 1607: 460393, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31376982

RESUMO

Characterization of silver-associated proteins is important to elucidate underlined mechanisms of silver-containing materials against microbes. Gel electrophoresis based methods are the most popular and basic strategy for the analysis of biomolecules, i.e., proteins and nucleic acids. It solely provides molecular weights of analytes. Extending the method from molecular weight to elemental composition is highly desired when investigating metal-containing molecules. Herein, a gel electrophoresis based method combining native sodium dodecyl sulfate-polyacrylamide gel electrophoresis (native SDS-PAGE), fluorescent staining, and inductively coupled plasma mass spectrometry (ICP-MS) strategy was developed for separation and detection of silver-associated proteins. Two home-made silver-labeled proteins, carbonic anhydrase and ovalbumin, were used for validation of the strategy performance. Silver-associated proteins in Pseudomonas aeruginosa and Staphylococcus aureus treated with silver nanoparticles were further characterized by this method. Some well-known and new proteins were identified to associate to silver in both P. aeruginosa and S. aureus, demonstrating the feasibility of the developed strategy. In conclusion, the current study provides a convenient method for readily identification of silver-associated proteins in biological samples.


Assuntos
Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Nanopartículas Metálicas/química , Pseudomonas aeruginosa/metabolismo , Prata/química , Espectrofotometria Atômica/métodos , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Anidrases Carbônicas/metabolismo , Fluorescência , Peso Molecular , Ovalbumina/metabolismo , Proteínas de Prata
16.
J Pharm Biomed Anal ; 175: 112785, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31352170

RESUMO

An antibiotic-affinity method was developed for analyzing Staphylococcus on the basis of the strong binding capability of daptomycin towards Gram-positive bacteria cellular membrane, as well as the selective lytic action of lysostaphin towards Staphylococcus. Daptomycin-modified magnetic beads were adopted to enrich Staphylococcus from sample matrix. Afterwards lysostaphin was adopted to lyse Staphylococcus, which can hydrolyze pentaglycine cross-linkers of peptidoglycan composing the cellular wall of Staphylococcus. The concentration of Staphylococcus was quantified by collecting the bioluminescent signal of the released intracellular adenosine triphosphate of the enriched Staphylococcus. Staphylococcus aureus (S. aureus) was analyzed as a model bacterium to study the feasibility of the proof-of-principle work. For bioluminescent analysis of S. aureus with the developed method, the linear range was 5.0 × 102-5.0 × 106 colony forming units mL-1, and the limit of detection was 3.8 × 102 colony forming units mL-1. The analytical procedure consisting of bacterial enrichment, cell lysis and signal collection can be accomplished within 20 min. Some common Gram-positive bacteria and Gram-negative bacteria all indicated very low interference to the analysis of the target bacterium. It has been successfully used to analyze S. aureus in milk as well as physiological saline injection, indicating its application potential for real samples.


Assuntos
Daptomicina/metabolismo , Lisostafina/metabolismo , Staphylococcus aureus/metabolismo , Animais , Antibacterianos/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Fenômenos Magnéticos , Leite/metabolismo , Leite/microbiologia
17.
Nat Commun ; 10(1): 3067, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296851

RESUMO

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Staphylococcus aureus/metabolismo , Zinco/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cátions Bivalentes/metabolismo , Histidina/genética , Histidina Quinase/química , Histidina Quinase/genética , Simulação de Dinâmica Molecular , Mutação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Regulon/genética , Staphylococcus aureus/genética , Tirosina/genética
18.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31308080

RESUMO

Macrophages are critical mediators of innate immunity and must be overcome for bacterial pathogens to cause disease. The Gram-positive bacterium Staphylococcus aureus produces virulence factors that impede macrophages and other immune cells. We previously determined that production of the metabolic cofactor lipoic acid by the lipoic acid synthetase, LipA, blunts macrophage activation. A ΔlipA mutant was attenuated during infection and was more readily cleared from the host. We hypothesized that bacterial lipoic acid synthesis perturbs macrophage antimicrobial functions and therefore hinders the clearance of S. aureus Here, we found that enhanced innate immune cell activation after infection with a ΔlipA mutant was central to attenuation in vivo, whereas a growth defect imparted by the lipA mutation made a negligible contribution to overall clearance. Macrophages recruited to the site of infection with the ΔlipA mutant produced larger amounts of bactericidal reactive oxygen species (ROS) and reactive nitrogen species (RNS) than those recruited to the site of infection with the wild-type strain or the mutant strain complemented with lipA ROS derived from the NADPH phagocyte oxidase complex and RNS derived from the inducible nitric oxide synthetase, but not mitochondrial ROS, were critical for the restriction of bacterial growth under these conditions. Despite enhanced antimicrobial immunity upon primary infection with the ΔlipA mutant, we found that the host failed to mount an improved recall response to secondary infection. Our data suggest that lipoic acid synthesis in S. aureus promotes bacterial persistence during infection through limitation of ROS and RNS generation by macrophages. Broadly, this work furthers our understanding of the intersections between bacterial metabolism and immune responses to infection.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Macrófagos Peritoneais/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Ácido Tióctico/biossíntese , Animais , Proteínas de Bactérias/metabolismo , Feminino , Interações Hospedeiro-Patógeno/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Masculino , Camundongos , Viabilidade Microbiana , Mutação , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Espécies Reativas de Nitrogênio/antagonistas & inibidores , Espécies Reativas de Nitrogênio/imunologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Ácido Tióctico/farmacologia
19.
PLoS Pathog ; 15(7): e1007971, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31344131

RESUMO

The ability of Staphylococcus aureus and other pathogens to consume glucose is critical during infection. However, glucose consumption increases the cellular demand for manganese sensitizing S. aureus to host-imposed manganese starvation. The current investigations were undertaken to elucidate how S. aureus copes with the need to consume glucose when metal-limited by the host. A critical component of host defense is production of the manganese binding protein calprotectin. S. aureus has two variants of phosphoglycerate mutase, one of which is manganese-dependent, GpmI, and another that is manganese-independent, GpmA. Leveraging the ability to impose metal starvation in culture utilizing calprotectin revealed that the loss of GpmA, but not GpmI, sensitized S. aureus to manganese starvation. Metabolite feeding experiments revealed that the growth defect of GpmA when manganese-starved was due to a defect in glycolysis and not gluconeogenesis. Loss of GpmA reduces the ability of S. aureus to cause invasive disease in wild type mice. However, GpmA was dispensable in calprotectin-deficient mice, which have defects in manganese sequestration, indicating that this isozyme contributes to the ability of S. aureus to overcome manganese limitation during infection. Cumulatively, these observations suggest that expressing a metal-independent variant enables S. aureus to consume glucose while mitigating the negative impact that glycolysis has on the cellular demand for manganese. S. aureus is not the only bacterium that expresses manganese-dependent and -independent variants of phosphoglycerate mutase. Similar results were also observed in culture with Salmonella enterica serovar Typhimurium mutants lacking the metal-independent isozyme. These similar observations in both Gram-positive and Gram-negative pathogens suggest that expression of metal-independent glycolytic isozymes is a common strategy employed by bacteria to survive in metal-limited environments, such as the host.


Assuntos
Metais/metabolismo , Fosfoglicerato Mutase/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Variação Genética , Glicólise , Isoenzimas/genética , Isoenzimas/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Manganês/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoglicerato Mutase/genética , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Virulência
20.
J Biomol NMR ; 73(5): 223-227, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31165320

RESUMO

Staphylococcus aureus hibernation promoting factor (SaHPF) is a 22,2 kDa protein which plays a crucial role in 100S Staphylococcus aureus ribosome formation during stress. SaHPF consists of N-terminal domain (NTD) that prevents proteins synthesis by binding to the 30S subunit at the P- and A-sites, connected through a flexible linker with a C-terminal domain (CTD) that keeps ribosomes in 100S form via homodimerization. Recently obtained 100S ribosome structure of S. aureus by cryo-EM shown that SaHPF-NTD bound to the ribosome active sites, however due to the absence of SaHPF-NTD structure it was modeled by homology with the E. coli hibernation factors HPF and YfiA. In present paper we have determined the solution structure of SaHPF-NTD by high-resolution NMR spectroscopy which allows us to increase structural knowledge about HPF structure from S. aureus.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo
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