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1.
Food Chem ; 337: 127776, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777574

RESUMO

Staphylococcus aureus is a Gram-positive bacterium responsible for a number of diseases and has demonstrated resistance to conventional antibiotics. This study aimed to evaluate the antibacterial activity of eugenol and its derivatives allylbenzene, 4-allylanisole, isoeugenol and 4-allyl-2,6-dimethoxyphenol against the S. aureus NorA efflux pump (EP) in association with norfloxacin and ethidium bromide. The antibacterial activity of the compounds was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC). A reduction in the MIC of ethidium bromide (a substrate for several efflux pumps) or norfloxacin was used as a parameter of EP inhibition. Molecular modeling studies were used to predict the 3D structure and analyze the interaction of selected compounds with the binding pocket of the NorA efflux pump. Except for 4-allylanisole and allylbenzene, the compounds presented clinically effective antibacterial activity. When associated with norfloxacin against the SA 1199B strain, 4-allyl-2,6-dimethoxyphenol eugenol and isoeugenol caused significant reduction in the MIC of the antibiotic, demonstrating synergistic effects. Similar effects were observed when 4-allyl-2,6-dimethoxyphenol, allylbenzene and isoeugenol were associated with ethidium bromide. Together, these findings indicate a potential inhibition of the NorA pump by eugenol and its derivatives. This in vitro evidence was corroborated by docking results demonstrating favorable interactions between 4-allyl-2,6-dimetoxypheno and the NorA pump mediated by hydrogen bonds and hydrophobic interactions. In conclusion, eugenol derivatives have the potential to be used in antibacterial drug development in strains carrying the NorA efflux pump.


Assuntos
Proteínas de Bactérias/metabolismo , Eugenol/análogos & derivados , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Staphylococcus aureus/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Etídio/farmacologia , Eugenol/metabolismo , Eugenol/farmacologia , Ligação de Hidrogênio , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Norfloxacino/farmacologia , Staphylococcus aureus/efeitos dos fármacos
2.
PLoS Genet ; 16(12): e1009282, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33378356

RESUMO

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ligases/genética , Staphylococcus aureus/genética , Estresse Fisiológico , Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Staphylococcus aureus/metabolismo
3.
Shokuhin Eiseigaku Zasshi ; 61(4): 138-142, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33012768

RESUMO

Staphylococcus aureus food poisoning is caused by the intoxication of staphylococcal enterotoxin (SE) produced in foods. Staphylococcal food poisoning is mostly due to staphylococcal enterotoxin type A (SEA) among SEs. There have been many studies on the growth and SEA production of S. aureus in various foods, but few studies in bread. Thus, the SEA production by S. aureus in dough during fermentation and the SEA inactivation in dough during baking were studied in the normal production processes of bread in this study. No growth of S. aureus or SEA production in dough, whose total weight was about 470 g, was observed during the fermentation at 25 and 35℃ for four hr, suggesting that the risk of SEA production in dough during fermentation under these conditions would be negligible. Any SEA injected at 6.0 and 0.56 ng/g in dough could not be detected after 20 and 10 min of baking at 200℃, respectively. These results showed that the baking process, which was completed in 25 min, was enough to inactivate SEA at those doses of SEA in the dough. The results on the production and inactivation of SEA in dough during the production processes in this study would be useful information on microbiological food safety of bread making.


Assuntos
Pão , Enterotoxinas , Viabilidade Microbiana , Staphylococcus aureus , Pão/microbiologia , Enterotoxinas/metabolismo , Temperatura Alta , Humanos , Intoxicação Alimentar Estafilocócica/microbiologia , Intoxicação Alimentar Estafilocócica/prevenção & controle , Staphylococcus aureus/metabolismo
4.
J Med Microbiol ; 69(10): 1253-1261, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32909934

RESUMO

Introduction. Staphylococcus aureus is a skin and mucous commensal bacterium of warm-blooded animals. In humans, the nose is the main ecological niche of S. aureus, and nasal carriage is a risk factor for developing an endogenous infection. S. aureus nasal colonization is a multifactorial process, involving inter-species interactions among the nasal microbiota.Aims. The objectives of this study were to characterize the microbiota of carriers and non-carriers of S. aureus and to demonstrate the importance of inter-species relationships in the adhesion of S. aureus, a key step in nasal colonization.Methodology. First, we characterized the nasal microbiota from 30 S. aureus carriers and non-carriers by a culturomic approach. We then evaluated the adhesion of S. aureus, first alone and then along with other bacteria of the nasal microbiota. To do that, we used an in vitro model to measure the interactions among bacteria in the presence of epithelial cells.Results. Analysis of the nasal microbiota of the carriers and non-carriers of S. aureus made it possible to observe that each microbiota has specific features in terms of composition. However, this composition differs significantly between carriers and non-carriers mainly through two bacterial groups: coagulase-negative staphylococci and corynebacteria. In a second part, adhesion of S. aureus to epithelial cells showed competition between S. aureus and these bacteria, suggesting a limitation of nasal colonization by S. aureus.Conclusion. These findings demonstrate the existence of a negative correlation between S. aureus and other species which inhibits adhesion and could limit nasal colonization.


Assuntos
Aderência Bacteriana/fisiologia , Mucosa Nasal/metabolismo , Staphylococcus aureus/metabolismo , Adulto , Bactérias , Portador Sadio/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Humanos , Masculino , Microbiota , Cavidade Nasal/microbiologia , Mucosa Nasal/microbiologia , Nariz/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade
5.
Nat Commun ; 11(1): 3888, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753666

RESUMO

First proposed as antimicrobial agents, histones were later recognized for their role in condensing chromosomes. Histone antimicrobial activity has been reported in innate immune responses. However, how histones kill bacteria has remained elusive. The co-localization of histones with antimicrobial peptides (AMPs) in immune cells suggests that histones may be part of a larger antimicrobial mechanism in vivo. Here we report that histone H2A enters E. coli and S. aureus through membrane pores formed by the AMPs LL-37 and magainin-2. H2A enhances AMP-induced pores, depolarizes the bacterial membrane potential, and impairs membrane recovery. Inside the cytoplasm, H2A reorganizes bacterial chromosomal DNA and inhibits global transcription. Whereas bacteria recover from the pore-forming effects of LL-37, the concomitant effects of H2A and LL-37 are irrecoverable. Their combination constitutes a positive feedback loop that exponentially amplifies their antimicrobial activities, causing antimicrobial synergy. More generally, treatment with H2A and the pore-forming antibiotic polymyxin B completely eradicates bacterial growth.


Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Estruturas Cromossômicas/efeitos dos fármacos , Histonas/metabolismo , Prótons , Animais , Estruturas Cromossômicas/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Imunidade Inata , Mamíferos , Polimixina B/farmacologia , Análise de Sequência de RNA , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(34): 20836-20847, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32769205

RESUMO

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.


Assuntos
Staphylococcus aureus/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Família Multigênica/genética , Transporte Proteico/genética , Proteômica , Infecções Estafilocócicas/microbiologia , Toxinas Biológicas/metabolismo , Sistemas de Secreção Tipo VII/fisiologia , Virulência/genética , Peixe-Zebra/microbiologia
7.
Nat Commun ; 11(1): 4097, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796861

RESUMO

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.


Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus/citologia , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Microbiologia , Imagem com Lapso de Tempo
8.
BMC Infect Dis ; 20(1): 621, 2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831057

RESUMO

BACKGROUND: We aimed to describe an outbreak of cutaneous abscesses caused by Panton-Valentine leukocidin (PVL)-producing methicillin-susceptible Staphylococcus aureus (MSSA) among gold mine workers. METHODS: In February 2018, we retrospectively reviewed a random sample of 50 medical records from 243 cases and conducted face-to-face interviews using a structured questionnaire. Pus aspirates were sent to the National Institute for Communicable Diseases from prospectively-identified cases (November 2017-March 2018). Nasopharyngeal swabs were collected during a colonisation survey in February 2018. Staphylococcus aureus isolates were screened with a conventional PCR for lukS/F-PV. Pulsed-field gel electrophoresis (PFGE) was performed to determine the genetic relatedness among the isolates. A sample of isolates were selected for whole genome sequencing (WGS). We conducted an assessment on biological risks associated with mining activities. RESULTS: From January 2017 to February 2018, 10% (350/3582) of mine workers sought care for cutaneous abscesses. Forty-seven medical files were available for review, 96% were male (n = 45) with a mean age of 43 years (SD = 7). About 52% (24/46) were involved in stoping and 28% (13/47) worked on a particular level. We cultured S. aureus from 79% (30/38) of cases with a submitted specimen and 14% (12/83) from colonisation swabs. All isolates were susceptible to cloxacillin. Seventy-one percent of S. aureus isolates (30/42) were PVL-PCR-positive. Six PFGE clusters were identified, 57% (21/37) were closely related. WGS analysis found nine different sequence types. PFGE and WGS analysis showed more than one cluster of S. aureus infections involving closely related isolates. Test reports for feed and product water of the mine showed that total plate counts were above the limits of 1000 cfu/ml, coliform counts > 10 cfu/100 ml and presence of faecal coliforms. Best practices were poorly implemented as some mine workers washed protective clothing with untreated water and hung them for drying at the underground surface. CONCLUSIONS: PVL-producing MSSA caused an outbreak of cutaneous abscesses among underground workers at a gold mining company. To our knowledge, no other outbreaks of PVL-producing S. aureus involving skin and soft tissue infections have been reported in mining facilities in South Africa. We recommend that worker awareness of infection prevention and control practices be strengthened.


Assuntos
Abscesso/microbiologia , Dermatopatias/epidemiologia , Infecções dos Tecidos Moles/epidemiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/patogenicidade , Adulto , Toxinas Bacterianas/metabolismo , Surtos de Doenças , Eletroforese em Gel de Campo Pulsado , Exotoxinas/metabolismo , Feminino , Ouro , Humanos , Leucocidinas/metabolismo , Masculino , Meticilina/farmacologia , Pessoa de Meia-Idade , Mineradores , Estudos Retrospectivos , Dermatopatias/microbiologia , Infecções dos Tecidos Moles/microbiologia , África do Sul/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
9.
Nat Commun ; 11(1): 3526, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665571

RESUMO

Gene tandem amplifications are thought to drive bacterial evolution, but they are transient in the absence of selection, making their investigation challenging. Here, we analyze genomic sequences of Staphylococcus aureus USA300 isolates from the same geographical area to identify variations in gene copy number, which we confirm by long-read sequencing. We find several hotspots of variation, including the csa1 cluster encoding lipoproteins known to be immunogenic. We also show that the csa1 locus expands and contracts during bacterial growth in vitro and during systemic infection of mice, and recombination creates rapid heterogeneity in initially clonal cultures. Furthermore, csa1 copy number variants differ in their immunostimulatory capacity, revealing a mechanism by which gene copy number variation can modulate the host immune response.


Assuntos
Genoma Bacteriano/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Animais , Evolução Biológica , Genótipo , Camundongos , Fenótipo , Staphylococcus aureus/patogenicidade
10.
PLoS Biol ; 18(7): e3000747, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644995

RESUMO

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Staphylococcus aureus/metabolismo , Biblioteca Gênica , Engenharia Genética , Loci Gênicos , Genoma Humano , Células HEK293 , Humanos , Nucleotídeos/genética , Fenótipo , Reprodutibilidade dos Testes , Ativação Transcricional/genética
11.
Appl Environ Microbiol ; 86(16)2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503913

RESUMO

Triclocarban (TCC), a formerly used disinfectant, kills bacteria via an unknown mechanism of action. A structural hallmark is its N,N'-diaryl urea motif, which is also present in other antibiotics, including the recently reported small molecule PK150. We show here that, like PK150, TCC exhibits an inhibitory effect on Staphylococcus aureus menaquinone metabolism via inhibition of the biosynthesis protein demethylmenaquinone methyltransferase (MenG). However, the activity spectrum (MIC90) of TCC across a broad range of multidrug-resistant staphylococcus and enterococcus strains was much narrower than that of PK150. Accordingly, TCC did not cause an overactivation of signal peptidase SpsB, a hallmark of the PK150 mode of action. Furthermore, we were able to rule out inhibition of FabI, a confirmed target of the diaryl ether antibiotic triclosan (TCS). Differences in the target profiles of TCC and TCS were further investigated by proteomic analysis, showing complex but rather distinct changes in the protein expression profile of S. aureus Downregulation of the arginine deiminase pathway provided additional evidence for an effect on bacterial energy metabolism by TCC.IMPORTANCE TCC's widespread use as an antimicrobial agent has made it a ubiquitous environmental pollutant despite its withdrawal due to ecological and toxicological concerns. With its antibacterial mechanism of action still being unknown, we undertook a comparative target analysis between TCC, PK150 (a recently discovered antibacterial compound with structural resemblance to TCC), and TCS (another widely employed chlorinated biphenyl antimicrobial) in the bacterium Staphylococcus aureus We show that there are distinct differences in each compound's mode of action, but also identify a shared target between TCC and PK150, the interference with menaquinone metabolism by inhibition of MenG. The prevailing differences, however, which also manifest in a remarkably better broad-spectrum activity of PK150, suggest that even high levels of TCC or TCS resistance observed by continuous environmental exposure may not affect the potential of PK150 or related N,N'-diaryl urea compounds as new antibiotic drug candidates against multidrug-resistant infections.


Assuntos
Proteínas de Bactérias/genética , Carbanilidas/farmacologia , Desinfetantes/farmacologia , Enterococcus/efeitos dos fármacos , Metiltransferases/genética , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Enterococcus/genética , Enterococcus/metabolismo , Metiltransferases/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
12.
Arch Microbiol ; 202(8): 2189-2196, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32524179

RESUMO

Three hydrocarbon degrading microorganisms were isolated from the soil samples collected from the storage sites of low viscosity spindle oil containers and identified on the basis of morphological and biochemical characteristics as Aeromonas hydrophila, Bacillus subtilis and Staphylococcus aureus. The study has revealed high ability of these microorganisms for oil biodegradation. The results have indicated that all isolates had the potential to breakdown the hydrocarbon. The most efficient bacteria among these examined was Aeromonas hydrophila which biodegraded almost all tested hydrocarbon giving a treatment percentage of 98% within 30 days which was considered as the perfect period for degradation. Also, a small scale was designed to treat the spindle oil with the using of oxidation process and all the tested organic materials were biodegraded in a treatment percentage of 100% within retention time of 20 days.


Assuntos
Bactérias/metabolismo , Biodegradação Ambiental , Petróleo/microbiologia , Microbiologia do Solo , Poluentes do Solo/metabolismo , Aeromonas hydrophila/metabolismo , Bacillus subtilis/metabolismo , Bactérias/isolamento & purificação , Hidrocarbonetos/metabolismo , Petróleo/metabolismo , Poluição por Petróleo , Staphylococcus aureus/metabolismo , Viscosidade
13.
PLoS One ; 15(5): e0233485, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470050

RESUMO

Antimicrobial resistance is a growing global health and economic concern. Current antimicrobial agents are becoming less effective against common bacterial infections. We previously identified pyrrolocins A and C, which showed activity against a variety of Gram-positive bacteria. Structurally similar compounds, known as pyrrolidinediones (e.g., TA-289, equisetin), also display antibacterial activity. However, the mechanism of action of these compounds against bacteria was undetermined. Here, we show that pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase (ACC), the first step in fatty acid synthesis. We used transcriptomic data, metabolomic analysis, fatty acid rescue and acetate incorporation experiments to show that a major mechanism of action of the pyrrolidinediones is inhibition of fatty acid biosynthesis, identifying ACC as the probable molecular target. This hypothesis was further supported using purified proteins, demonstrating that biotin carboxylase is the inhibited component of ACC. There are few known antibiotics that target this pathway and, therefore, we believe that these compounds may provide the basis for alternatives to current antimicrobial therapy.


Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Pirrolidinonas/farmacologia , Tetra-Hidronaftalenos/farmacologia , Acetil-CoA Carboxilase/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Domínio Catalítico/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/biossíntese , Perfilação da Expressão Gênica , Bactérias Gram-Positivas/crescimento & desenvolvimento , Humanos , Metabolômica , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(20): 10989-10999, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32354997

RESUMO

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas , Sequência de Bases , Biofilmes , Domínio Catalítico , Modelos Animais de Doenças , Endocardite , Enterotoxinas , Feminino , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/metabolismo , Masculino , Modelos Moleculares , Mutação , Oxirredução , Domínios Proteicos , Coelhos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Sepse , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Superantígenos , Thermotoga maritima , Virulência/genética , Virulência/fisiologia
15.
Sci Rep ; 10(1): 8467, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439871

RESUMO

Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches.


Assuntos
Proteínas de Bactérias/metabolismo , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Proteoma/análise , Proteoma/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Bovinos , Humanos , Ovinos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/isolamento & purificação
16.
BMC Infect Dis ; 20(1): 354, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32429852

RESUMO

BACKGROUND: Mediastinitis caused by hematogenous spread of an infection is rare. We report the first known case of community-acquired mediastinitis from hematogenous origin in an immunocompetent adult. This rare invasive infection was due to Panton-Valentine Leucocidin-producing (PVL+) methicillin-susceptible Staphylococcus aureus (MSSA). CASE PRESENTATION: A 22-year-old obese man without other medical history was hospitalized for febrile precordial chest pain. He reported a cutaneous back abscess 3 weeks before. CT-scan was consistent with mediastinitis and blood cultures grew for a PVL+ MSSA. Intravenous clindamycin (600 mg t.i.d) and cloxacillin (2 g q.i.d.), secondary changed for fosfomycin (4 g q.i.d.) because of a related toxidermia, was administered. Surgical drainage was performed and confirmed the presence of a mediastinal abscess associated with a fistula between the mediastinum and right pleural space. All local bacteriological samples also grew for PVL+ MSSA. In addition to clindamycin, intravenous fosfomycin was switched to trimethoprim-sulfamethoxazole after 4 weeks for a total of 10 weeks of antibiotics. CONCLUSIONS: We present the first community-acquired mediastinitis of hematogenous origin with PVL+ MSSA. Clinical evolution was favorable after surgical drainage and 10 weeks of antibiotics. The specific virulence of MSSA PVL+ strains played presumably a key role in this rare invasive clinical presentation.


Assuntos
Toxinas Bacterianas/análise , Infecções Comunitárias Adquiridas/diagnóstico , Exotoxinas/análise , Imunocompetência , Leucocidinas/análise , Mediastinite/diagnóstico , Mediastinite/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/metabolismo , Abscesso/tratamento farmacológico , Abscesso/microbiologia , Abscesso/cirurgia , Antibacterianos/uso terapêutico , Anti-Infecciosos Urinários/uso terapêutico , Clindamicina/uso terapêutico , Infecções Comunitárias Adquiridas/tratamento farmacológico , Drenagem , Humanos , Masculino , Mediastinite/tratamento farmacológico , Mediastinite/imunologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Resultado do Tratamento , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico , Adulto Jovem
17.
Proc Natl Acad Sci U S A ; 117(22): 12394-12401, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414924

RESUMO

The bacterial pathogen Staphylococcus aureus is capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs. S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasive S. aureus infection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasive S. aureus infection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival of S. aureus mutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered that S. aureus requires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, and S. aureus instead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition by S. aureus Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.


Assuntos
Ácido Aspártico/biossíntese , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Nutrientes/metabolismo , Osteomielite/metabolismo , Osteomielite/microbiologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética
18.
Nat Commun ; 11(1): 2211, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371860

RESUMO

Microbial biofilm formation on indwelling medical devices causes persistent infections that cannot be cured with conventional antibiotics. To address this unmet challenge, we engineer tunable active surface topographies with micron-sized pillars that can beat at a programmable frequency and force level in an electromagnetic field. Compared to the flat and static controls, active topographies with the optimized design prevent biofilm formation and remove established biofilms of uropathogenic Escherichia coli (UPEC), Pseudomonas aeruginosa, and Staphylococcus aureus, with up to 3.7 logs of biomass reduction. In addition, the detached biofilm cells are found sensitized to bactericidal antibiotics to the level comparable to exponential-phase planktonic cells. Based on these findings, a prototype catheter is engineered and found to remain clean for at least 30 days under the flow of artificial urine medium, while the control catheters are blocked by UPEC biofilms within 5 days.


Assuntos
Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia , Escherichia coli Uropatogênica/fisiologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Biofilmes/efeitos dos fármacos , Biomassa , Campos Eletromagnéticos , Testes de Sensibilidade Microbiana/métodos , Infecções Relacionadas à Prótese/microbiologia , Infecções Relacionadas à Prótese/prevenção & controle , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Fatores de Tempo , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/metabolismo
19.
Nat Commun ; 11(1): 1656, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245971

RESUMO

For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here we describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections.


Assuntos
Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenvolvimento de Medicamentos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Subunidades Ribossômicas
20.
Exp Eye Res ; 193: 107994, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32147399

RESUMO

Staphylococcus aureus is a common bacterial isolate from cases of microbial keratitis. The virulence factors that contribute to its pathogenicity during this disease have not been fully resolved. The aim of the current study was to examine the effects of the extracellular protease Staphopain A on corneal virulence. Two strains were used, one Staph 38 that gives a high pathology score during keratitis and a less virulent strain ATCC 8325-4. The effect of inhibition of Staphopain by general or specific protease inhibitors on adhesion of strains to fibronectin-coated glass or PMMA was determined. This was followed by an analysis of the effect of Staphopain A on the ability of the bacteria to adhere to and invade corneal epithelial cells. Finally, the effect of inhibiting Staphopain A on pathogenesis in a mouse model of keratitis was studied. Staphopain A increased the adhesion of strains to fibronectin-coated substrata and inhibition of Staphopain A reduced adhesion. The inhibition of Staphopain A by staphostatin A significantly decreased both association with and invasion into human corneal epithelial cells by 15-fold for strain Saur38. Inhibition of Staphopain A significantly reduced the pathology associated with S. aureus keratitis, reducing the infecting numbers of bacteria from 1.8x105 to <1x104 cells/cornea (p ≤ 0.001), significantly reducing the corneal pathology score (p ≤ 0.038) and reducing the numbers of infiltrating PMNs. This study shows that Staphopain increases adhesion and invasion of corneal cells due to increasing fibronectin binding and its inhibition has a significant impact on pathogenicity of S. aureus during keratitis.


Assuntos
Cisteína Endopeptidases/metabolismo , Infecções Oculares Bacterianas/microbiologia , Ceratite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Modelos Animais de Doenças , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/patologia , Humanos , Ceratite/metabolismo , Ceratite/patologia , Masculino , Camundongos , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/patologia
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