Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Mol Microbiol ; 2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34626146

RESUMO

In Staphylococcus aureus, the two-component system SaeRS is responsible for regulating various virulence factors essential for the success of this pathogen. SaeRS can be stimulated by neutrophil-derived products but has also recently been shown to be inactivated by the presence of free fatty acids. A mechanism for how fatty acids negatively impacts SaeRS has not been described. We found that unsaturated fatty acids, as well as fatty acids not commonly found in Staphylococcal membranes, prevent the activation of SaeRS at a lower concentration than their saturated counterparts. These fatty acids can negatively impact SaeRS without altering the respiratory capacity of the bacterium. To uncover a potential mechanism for how fatty acids impact SaeRS function/activity, we utilized a naturally occurring point mutation found in S. aureus as well as chimeric SaeS proteins. Using these tools, we identified that the native transmembrane domains of SaeS dictate the transcriptional response to fatty acids in S. aureus. Our data support a model where free fatty acids alter the activity of the two-component system SaeRS directly through the sensor kinase SaeS and is dependent on the transmembrane domains of the protein.

2.
Methods Mol Biol ; 2341: 25-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264457

RESUMO

Many strains of Staphylococcus aureus produce a variety of cytolysins that target many different cell types to both fight the immune system and acquire nutrients. This includes hemolysins which destroy erythrocytes and are well studied virulence factors. Traditionally, hemolysin activity is measured on blood agar plates due to the simplicity of the assay. While this is telling, it cannot encapsulate the full story because S. aureus is known to behave differently in broth and on agar. Furthermore, plate-based assays are primarily semiquantitative and often a more accurate determination of hemolytic potential is needed to discern differences between strains. Here, we describe a method to quantify hemolysin activity from broth or similarly grown cells.


Assuntos
Eritrócitos/fisiologia , Proteínas Hemolisinas/análise , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Proteínas Hemolisinas/metabolismo , Hemólise , Humanos , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/análise , Fatores de Virulência/metabolismo
3.
Methods Mol Biol ; 2341: 133-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264469

RESUMO

The use of cultured mammalian cells, whether immortalized cell lines or primary cells, is a well-known technique used as a substitute or prescreen for in vivo virulence potential of bacterial pathogens. This technique is also a way to examine host-pathogen interactions in a less complex environment compared to that found in whole animals. To this end, macrophage infection assays have become a key technique for studying the molecular mechanisms by which bacteria interact with the host. Herein, this chapter describes both how to produce macrophages from mouse bone marrow and the subsequent infection assays.


Assuntos
Técnicas de Cocultura/métodos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Animais , Linhagem Celular , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Células RAW 264.7
4.
Infect Immun ; 88(8)2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32513856

RESUMO

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.


Assuntos
Proteínas de Bactérias/imunologia , Metaloendopeptidases/imunologia , Fosfotransferases (Aceptor do Grupo Carboxila)/imunologia , Serina Endopeptidases/imunologia , Úlcera Cutânea/imunologia , Infecções Cutâneas Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Quimiocina CCL4/genética , Quimiocina CCL4/imunologia , Feminino , Regulação da Expressão Gênica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-1alfa/genética , Interleucina-1alfa/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Fosfotransferases (Aceptor do Grupo Carboxila)/deficiência , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Pele/imunologia , Pele/microbiologia , Pele/patologia , Úlcera Cutânea/genética , Úlcera Cutânea/microbiologia , Úlcera Cutânea/patologia , Infecções Cutâneas Estafilocócicas/genética , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologia
5.
Infect Immun ; 87(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30885928

RESUMO

To persist within the host and cause disease, Staphylococcus aureus relies on its ability to precisely fine-tune virulence factor expression in response to rapidly changing environments. During an unbiased transposon mutant screen, we observed that disruption of a two-gene operon, yjbIH, resulted in decreased levels of pigmentation and aureolysin (Aur) activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is predominantly responsible for the observed yjbIH mutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression of crtOPQMN and aur Previous studies found that YjbH targets the disulfide- and oxidative stress-responsive regulator Spx for degradation by ClpXP. The absence of yjbH or yjbI resulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in the yjbH and yjbI mutant strains. Decreased levels of pigmentation and aureolysin (Aur) activity in the yjbH mutant were found to be Spx dependent. Lastly, we used a murine sepsis model to determine the effect of the yjbIH deletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identified changes in pigmentation and protease activity in response to YjbIH and are the first to have shown a role for these proteins during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxirredutases/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/genética , Feminino , Humanos , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óperon , Oxirredutases/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo
6.
Infect Immun ; 87(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30420450

RESUMO

Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.


Assuntos
Proteínas de Bactérias , Óxido Nítrico Sintase/metabolismo , Proteínas Repressoras , Staphylococcus aureus , Virulência/fisiologia , Proteínas de Bactérias/genética , Células Cultivadas , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutação , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Estresse Oxidativo/fisiologia , Proteínas Repressoras/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Transcrição Genética , Virulência/genética
7.
Microbiology (Reading) ; 165(2): 197-207, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30566075

RESUMO

The human pathogen Staphylococcus aureus produces saturated fatty acids, but can incorporate both exogenous saturated and unsaturated fatty acids into its lipid membrane. S. aureus encounters unsaturated fatty acids in the host skin where they serve as an innate immune defence due to their toxicity. Previously, we identified a fatty acid kinase in S. aureus that is necessary for the utilization of exogenous fatty acids. The goal of this study was to determine the effects of fatty acids on mutants deficient in the exogenous fatty acid utilization machinery. We have demonstrated that mutants lacking a functional fatty acid kinase (fakA) or both fatty acid carrier proteins (fakB1 fakB2) are more resistant to unsaturated fatty acids. Previous studies suggested a role for ammonia-producing enzymes in resistance to unsaturated fatty acids, but these enzymes do not contribute to the resistance of the fakA mutant, despite increased urease transcription and protein activity in the mutant. Additionally, while pigment is altered in mutants unable to use exogenous fatty acids, staphyloxanthin does not contribute to fatty acid resistance of an fakA mutant. Because exposure to unsaturated fatty acids probably initiates a stress response, we investigated the role of the alternative sigma factor σB and determined if it is necessary for the fatty acid resistance observed in the fakA mutant. Collectively, this study demonstrates that the inability to incorporate unsaturated fatty acids leads to increased resistance to those fatty acids, and that resistance requires a σB stress response.


Assuntos
Vias Biossintéticas/genética , Ácidos Graxos Insaturados/toxicidade , Ácidos Graxos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Bactérias/genética , Ácidos Graxos Insaturados/metabolismo , Mutação , Fosfotransferases/genética , Fator sigma/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...