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1.
Microbiol Spectr ; 10(4): e0085322, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35938792

RESUMO

The emergence of each novel SARS-CoV-2 variant of concern (VOC) requires investigation of its potential impact on the performance of diagnostic tests in use, including antigen-detecting rapid diagnostic tests (Ag-RDTs). Although anecdotal reports have been circulating that the newly emerged Omicron-BA.1 variant is in principle detectable by Ag-RDTs, few data on sensitivity are available. We have performed (i) analytical sensitivity testing with cultured virus in eight Ag-RDTs and (ii) retrospective testing in duplicates with clinical samples from vaccinated individuals with Omicron-BA.1 (n = 59) or Delta (n = 54) breakthrough infection on seven Ag-RDTs. Overall, in our analytical study we have found heterogenicity between Ag-RDTs for detecting Omicron-BA.1. When using cultured virus, we observed a trend toward lower endpoint sensitivity for Omicron-BA.1 detection than for earlier circulating SARS-CoV-2 and the other VOCs. In our retrospective study, the detection of Delta and Omicron-BA.1 was assessed in a comparable set of stored clinical samples using seven Ag-RDTs. Four hundred ninety-seven of all 826 tests (60.17%) performed on Omicron-BA.1 samples were positive, compared to 489/756 (64.68%) for Delta samples. In the analytical study, the sensitivity for both Omicron-BA.1 and Delta between the Ag-RDTs was variable. All seven Ag-RDTs showed comparable sensitivities to detect Omicron-BA.1 and Delta in the retrospective study. IMPORTANCE Sensitivity for detecting Omicron-BA.1 shows high heterogenicity between Ag-RDTs, necessitating a careful consideration when using these tests to guide infection prevention measures. Analytical and retrospective testing is a proxy and timely solution to generate rapid performance data, but it is not a replacement for clinical evaluations, which are urgently needed. Biological and technical reasons for detection failure by some Ag-RDTs need to be further investigated.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Estudos Retrospectivos , SARS-CoV-2/genética , Sensibilidade e Especificidade
2.
Genes (Basel) ; 12(11)2021 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-34828356

RESUMO

Among clinically relevant lineages of Staphylococcus aureus, the lineage or clonal complex 398 (CC398) is of particular interest. Strains from this lineage were only described as livestock colonizers until 2007. Progressively, cases of infection were reported in humans in contact with farm animals, and now, CC398 isolates are increasingly identified as the cause of severe infections even in patients without any contact with animals. These observations suggest that CC398 isolates have spread not only in the community but also in the hospital setting. In addition, several recent studies have reported that CC398 strains are evolving towards increased virulence and antibiotic resistance. Identification of the origin and emergence of this clonal complex could probably benefit future large-scale studies that aim to detect sources of contamination and infection. Current evidence indicates that the evolution of CC398 strains towards these phenotypes has been driven by the acquisition of prophages and other mobile genetic elements. In this short review, we summarize the main knowledge of this major lineage of S. aureus that has become predominant in the human clinic worldwide within a single decade.


Assuntos
DNA Bacteriano/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Animais , Animais Domésticos/microbiologia , Evolução Molecular , Humanos , Sequências Repetitivas Dispersas , Vigilância da População , Prófagos/genética , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , Staphylococcus aureus/virologia
3.
Front Microbiol ; 10: 742, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31105650

RESUMO

Until 2007, Staphylococcus aureus from clonal complex 398 (CC398) was exclusively associated with livestock species and companion animals. Recently, several studies described the emergence of S. aureus CC398 as etiologies of severe infections in humans living in an animal-free environment. Recent sequencing efforts showed that the mobile genetic elements found in CC398 isolates were specific for each population and enabled differentiation of strains responsible for asymptomatic colonization from strains involved in bloodstream infections. We mobilized prophages from a human CC398 isolate and introduced them into two naïve ancestral isolates devoid of prophages that exclusively colonize animals. These lysogenized ancestral CC398 isolates acquired features related to virulence, such as an increased capacity to adhere to human extracellular matrix proteins and the ability to invade and survive within non-phagocytic cells. Pathogenicity of several clinical isolates from the CC398 lineage as well as ancestral and in vitro lysogenized ancestral counterparts was assessed in a model of infectious endocarditis in rats. Natural and artificial lysogens were not only more invasive than their prophage-free parent but also showed an increased capacity to multiply within aortic vegetations. This study identified prophages as mediators of bacterial virulence in a model of infectious endocarditis, probably through promotion of interaction with extracellular matrix components. Further studies are needed to identify mechanisms leading to promotion of intrinsic virulence.

4.
EMBO J ; 38(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30760492

RESUMO

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Glucose/deficiência , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Biofilmes/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Glucose/administração & dosagem , Redes e Vias Metabólicas , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Ribossomos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Edulcorantes/administração & dosagem , Transcriptoma
5.
Mol Microbiol ; 111(4): 1039-1056, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30636083

RESUMO

The intracellular redox environment of Staphylococcus aureus is mainly buffered by bacillithiol (BSH), a low molecular weight thiol. The identity of enzymes responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH) remains elusive. We examined YpdA, a putative bacillithiol reductase, for its role in maintaining intracellular redox homeostasis. The ypdA mutant showed increased levels of BSSB and a lower bacillithiol redox ratio vs. the isogenic parent, indicating a higher level of oxidative stress within the bacterial cytosol. We showed that YpdA consumed NAD(P)H; and YpdA protein levels were augmented in response to stress. Wild type strains overexpressing YpdA showed increased tolerance to oxidants and electrophilic agents. Importantly, YpdA overexpression in the parental strain caused an increase in BSH levels accompanied by a decrease in BSSB concentration in the presence of stress, resulting in an increase in bacillithiol redox ratio vs. the vector control. Additionally, the ypdA mutant exhibited decreased survival in human neutrophils (PMNs) as compared with the parent, while YpdA overexpression protected the resulting strain from oxidative stress in vitro and from killing by human neutrophils ex vivo. Taken together, these data present a new role for YpdA in S. aureus physiology and virulence through the bacillithiol system.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Quinases/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Células Cultivadas , Homeostase , Humanos , Mutação , Neutrófilos/microbiologia , Oxirredução , Proteínas Quinases/genética , Staphylococcus aureus/genética , Virulência
7.
Infect Immun ; 86(2)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29133345

RESUMO

Expression of virulence factors in Staphylococcus aureus is regulated by a wide range of transcriptional regulators, including proteins and small RNAs (sRNAs), at the level of transcription and/or translation. The sarA locus consists of three overlapping transcripts generated from three distinct promoters, all containing the sarA open reading frame (ORF). The 5' untranslated regions (UTRs) of these transcripts contain three separate regions ∼711, 409, and 146 nucleotides (nt) upstream of the sarA translation start, the functions of which remain unknown. Recent transcriptome-sequencing (RNA-Seq) analysis and subsequent characterization indicated that two sRNAs, teg49 and teg48, are processed and likely produced from the sarA P3 and sarA P1 transcripts of the sarA locus, respectively. In this report, we utilized a variety of sarA promoter mutants and cshA and rnc mutants to ascertain the contributions of these factors to the generation of teg49. We also defined the transcriptional regulon of teg49, including virulence genes not regulated by SarA. Phenotypically, teg49 did not impact biofilm formation or affect overall SarA expression significantly. Comparative analyses of RNA-Seq data between the wild-type, teg49 mutant, and sarA mutant strains indicated that ∼133 genes are significantly upregulated while 97 are downregulated in a teg49 deletion mutant in a sarA-independent manner. An abscess model of skin infection indicated that the teg49 mutant exhibited a reduced bacterial load compared to the wild-type S. aureus Overall, these results suggest that teg49 sRNA has a regulatory role in target gene regulation independent of SarA. The exact mechanism of this regulation is yet to be dissected.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Fatores de Virulência/biossíntese , Abscesso/microbiologia , Abscesso/patologia , Animais , Modelos Animais de Doenças , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Camundongos Endogâmicos BALB C , Regulon , Infecções Cutâneas Estafilocócicas/patologia , Transcrição Gênica , Virulência
8.
J Bacteriol ; 199(16)2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28559294

RESUMO

Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.

9.
BMC Genomics ; 18(1): 133, 2017 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-28166723

RESUMO

BACKGROUND: It has been suggested that prophages in the ST398 S. aureus clone are responsible for expanding ST398's spectrum of action and increasing its ability to cause human infections. We carried out the first characterization of the various prophages carried by 76 ST398 bloodstream infection (BSI) isolates obtained over 9 years of observation. RESULTS: Whole-genome sequencing of 22 representative isolates showed (1) the presence of the φ3-prophage and diverse genetic features typical of animal-associated isolates (i.e., SCCmec XI element, Tn916 transposon and non φ3-prophages) in a majority of BSI isolates, (2) one BSI isolate devoid of the φ3-prophage but otherwise similar to an animal-infecting isolate, (3) 35 prophages carrying numerous genes previously associated with virulence or immune evasion in animal models of staphylococcal infections. The analysis of prophage content in all 76 BSI isolates showed an increasing prevalence of polylysogeny over time. Overall, over the course of the last 10 years, the BSI isolates appear to have acquired increasing numbers of genetic features previously shown to contribute to bacterial adaptation and virulence in animal models of staphylococcal infections. CONCLUSIONS: We hypothesize that lysogeny has played a significant role in increasing the ability of the ST398 clone to cause infections in humans. Our findings highlight the risk that the ST398 lineage will increase its threat to public health by continuing to acquire virulence and/or multiple antibiotic-resistance genes from hospital-associated clones of Staphylococcus aureus.


Assuntos
Adaptação Fisiológica , Prófagos/fisiologia , Staphylococcus aureus/fisiologia , Staphylococcus aureus/virologia , Animais , Evolução Molecular , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Sequenciamento Completo do Genoma
10.
Infect Immun ; 84(3): 833-44, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26755161

RESUMO

The toxin MazFsa in Staphylococcus aureus is a sequence-specific endoribonuclease that cleaves the majority of the mRNAs in vivo but spares many essential mRNAs (e.g., secY mRNA) and, surprisingly, an mRNA encoding a regulatory protein (i.e., sarA mRNA). We hypothesize that some mRNAs may be protected by RNA-binding protein(s) from degradation by MazFsa. Using heparin-Sepharose-enriched fractions that hybridized to sarA mRNA on Northwestern blots, we identified among multiple proteins the DEAD box RNA helicase CshA (NWMN_1985 or SA1885) by mass spectroscopy. Purified CshA exhibits typical RNA helicase activities, as exemplified by RNA-dependent ATPase activity and unwinding of the DNA-RNA duplex. A severe growth defect was observed in the cshA mutant compared with the parent when grown at 25°C but not at 37°C. Activation of MazFsa in the cshA mutant resulted in lower CFU per milliliter accompanied by a precipitous drop in viability (∼40%) compared to those of the parent and complemented strains. NanoString analysis reveals diminished expression of a small number of mRNAs and 22 small RNAs (sRNAs) in the cshA mutant versus the parent upon MazFsa induction, thus implying protection of these RNAs by CshA. In the case of the sRNA teg049 within the sarA locus, we showed that the protective effect was likely due to transcript stability as revealed by reduced half-life in the cshA mutant versus the parent. Accordingly, CshA likely stabilizes selective mRNAs and sRNAs in vivo and as a result enhances S. aureus survival upon MazFsa induction during stress.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Helicases/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , RNA Helicases/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
11.
Front Microbiol ; 5: 652, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25538688

RESUMO

Until recently, Staphylococcus aureus from clonal complex (CC)398 were mostly described as colonizing asymptomatic raised pigs and pig-farmers. Currently, the epidemiology of the CC398 lineage is becoming more complex. CC398 human-adapted isolates are increasingly being identified in bloodstream infections in humans living in animal-free environments. In addition, CC398 isolates are increasingly responsible for invasive infections in various animals. CC398 isolates that colonize asymptomatic pigs and the isolates that infect humans living in animal-free environments (human-adapted isolates) both lack several clinically important S. aureus-associated virulence factors but differ on the basis of their prophage content. Recent findings have provided insight into the influence of a φMR11-like helper prophage on the ability of CC398 isolates to infect humans. To assess the recent spread of the CC398 lineage to various animal species and to investigate the links between the φMR11-like prophage and the emergence of CC398 isolates infecting animals, we studied 277 isolates causing infections in unrelated animals. The prevalence of CC398 isolates increased significantly between 2007 and 2013 (p < 0.001); 31.8% of the animal isolates harbored the φMR11-like prophage. High-density DNA microarray experiments with 37 representative infected-animal isolates positive for φMR11-like DNA established that most infected-animal isolates carried many genetic elements related to antimicrobial resistance and virulence genes, and a φ3 prophage encoding immune-modulating proteins and associated with animal-to-human jumps. Our findings suggest recent clonal expansion and dissemination of a new subpopulation of CC398 isolates, responsible for invasive infections in various animals, with a considerable potential to colonize and infect humans, probably greater than that of human-adapted CC398 isolates, justifying active surveillance.

12.
Genome Announc ; 2(2)2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24625873

RESUMO

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (positive blood cultures after ≥7 days) represents a challenging subset of invasive MRSA infections. The comparison of genome sequences of persistent (300-169) and resolving (301-188) MRSA bacteremia isolates with similar genetic background (sequence type 45 [ST45]) will help us to better understand underlying mechanisms of persistent MRSA bacteremia.

13.
Genome Announc ; 1(5)2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24072856

RESUMO

Staphylococcus aureus sequence type 398 (ST398) was originally associated with animal infections. We announce the complete genome sequences of two ST398 methicillin-susceptible S. aureus strains from the livestock environment. These genome sequences assist in the characterization of interesting ST398 features relying on host tropism and epidemiological settings.

14.
Genome Announc ; 1(5)2013 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-23990587

RESUMO

Sequence type 398 (ST398) Staphylococcus aureus was originally associated with animal infection. We announce the complete genome sequences of two ST398 methicillin-susceptible S. aureus strains of human origin, S94 and S100. The genome sequences assist in the characterization of interesting ST398 features related to host specificities.

15.
Genome Announc ; 1(5)2013 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-23990588

RESUMO

Sequence type 398 (ST398) Staphylococcus aureus was originally reported in livestock. We announce the complete genome sequence of an ST398 methicillin-susceptible S. aureus strain of animal origin, S123. The genome sequence reveals a wild-type genome that probably corresponds to an ancestral clone.

16.
Infect Genet Evol ; 18: 299-308, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23770143

RESUMO

Staphylococcus aureus clonal complex 398 is a livestock-associated pathogen that poses a worldwide threat because of its ability to colonize and infect both humans and animals. We used high-resolution whole-genome microarrays, prophage profiling, immune evasion cluster characterization and whole-genome sequencing to investigate the roles of prophages in the emerging human-adapted subpopulation of CC398 that has been associated with invasive infections in humans living in animal-free environments. We characterized one phage and two prophages specifically harbored by CC398 isolates belonging to the emerging subpopulation. We introduced the phage into permissive prophage-free isolates. We investigated the effects of lysogeny on the host ability to resist further phage infection and transformation, to acquire the capacity to invade human cells, and to express virulence factors encoded by prophages. We report evidence of a defective ϕMR11-like helper prophage, named StauST398-5pro, specifically associated with the emerging non-LA CC398 subpopulation. StauST398-5pro confers substantial protection against horizontal genetic transfer to its host. It interacts with a human-associated ß-converting prophage encoding immune-modulating proteins such that virulence genes are expressed during stress situations. Our findings provide insight into the role of phages in the expression of virulence and in the spread of genetic information among new host-adapted S. aureus isolates. We demonstrate that functional prophage elements can condition host specificity and confer new virulence traits on emerging intra-species clones of bacteria.


Assuntos
Prófagos/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/virologia , Zoonoses/microbiologia , Animais , Distribuição de Qui-Quadrado , Farmacorresistência Bacteriana , Células HEK293 , Especificidade de Hospedeiro , Humanos , Gado , Lisogenia , Análise de Sequência com Séries de Oligonucleotídeos , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Virulência
17.
RNA Biol ; 10(1): 157-65, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23229022

RESUMO

DEAD-box RNA helicases are present in almost all living organisms and participate in various processes of RNA metabolism. Bacterial proteins of this large family were shown to be required for translation initiation, ribosome biogenesis and RNA decay. The latter is primordial for rapid adaptation to changing environmental conditions. In particular, the RhlB RNA helicase from E. coli was shown to assist the bacterial degradosome machinery. Recently, the CshA DEAD-box proteins from Bacillus subtilis and Staphylococcus aureus were shown to interact with proteins that are believed to form the degradosome. S. aureus can cause life-threatening disease, with particular concern focusing on biofilm formation on catheters and prosthetic devices, since in this form the bacteria are almost impossible to eradicate both by the immune system and antibiotic treatment. This persistent state relies on the expression of surface encoded proteins that allow attachment to various surfaces, and contrasts with the dispersal mode of growth that relies on the secretion of proteins such as hemolysins and proteases. The switch between these two states is mainly mediated by the Staphylococcal cell density sensing system encoded by agr. We show that inactivation of the cshA DEAD-box gene results in dysregulation of biofilm formation and hemolysis through modulation of agr mRNA stability. Importantly, inactivation of the agrA gene in the cshA mutant background reverses the defect, indicating that cshA is genetically upstream of agr and that a delicate balance of agr mRNA abundance mediated through stability control by CshA is critical for proper expression of virulence factors.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Helicases DEAD-box/metabolismo , Percepção de Quorum/fisiologia , Staphylococcus aureus/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Biofilmes , Ativação Enzimática , Hemólise , Mutação , Fenótipo , RNA/metabolismo , Estabilidade de RNA , Transativadores/genética
18.
Future Microbiol ; 6(5): 513-32, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21585260

RESUMO

Staphylococcus aureus is a major human pathogen responsible for a wide diversity of infections ranging from localized to life threatening diseases. From 1961 and the emergence of methicillin-resistant S. aureus (MRSA), this bacterium has shown a particular capacity to survive and adapt to drastic environmental changes and since the beginning of the 1990s it has spread worldwide. Until recently, S. aureus was considered as the prototype of a nosocomial pathogen but it has now been recognized as an agent responsible for outbreaks in the community. Several recent reports suggest that the epidemiology of MRSA is changing. Understanding of pathogenicity, virulence and emergence of epidemic clones within MRSA populations is not clearly defined, despite several attempts to identify common molecular features between strains that share similar epidemiological and/or virulence behavior. These studies included: pattern profiling of bacterial adhesins, analysis of clonal complex groups, molecular genotyping and enterotoxin content analysis. To date, all approaches failed to find a correlation between molecular determinants and clinical outcomes. We hypothesize that the capacity of the bacterium to become more invasive or virulent is determined by genetics. The utilization of massively parallel methods of analysis is therefore ideal to study the contribution of genetics. Therefore, this article focuses on the entire genome including coding sequences as well as noncoding sequences. This high resolution approach allows the monitoring micro- and macroevolution of MRSA and identification of specific genomic markers of evolution of invasive or highly virulent phenotypes.


Assuntos
Antibacterianos/farmacologia , Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Meticilina/farmacologia , Epidemiologia Molecular , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/patogenicidade , Genoma Bacteriano/efeitos dos fármacos , Genótipo , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Virulência
19.
Nucleic Acids Res ; 39(13): 5597-610, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21421560

RESUMO

A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg(++) buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. coli.


Assuntos
Enzimas de Restrição do DNA/metabolismo , Staphylococcus aureus/enzimologia , Trifosfato de Adenosina/metabolismo , Colífagos/fisiologia , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Nucleotídeos de Desoxiadenina/metabolismo , Escherichia coli/genética , Metais/química , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Cloreto de Sódio/química , Especificidade por Substrato
20.
Proc Natl Acad Sci U S A ; 107(26): 11954-8, 2010 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-20547849

RESUMO

Staphylococcus aureus is an versatile pathogen that can cause life-threatening infections. Depending on the clinical setting, up to 50% of S. aureus infections are caused by methicillin-resistant strains (MRSA) that in most cases are resistant to many other antibiotics, making treatment difficult. The emergence of community-acquired MRSA drastically changed the picture by increasing the risk of MRSA infections. Horizontal transfer of genes encoding for antibiotic resistance or virulence factors is a major concern of multidrug-resistant S. aureus infections and epidemiology. We identified and characterized a type III-like restriction system present in clinical S. aureus strains that prevents transformation with DNA from other bacterial species. Interestingly, our analysis revealed that some clinical MRSA strains are deficient in this restriction system, and thus are hypersusceptible to the horizontal transfer of DNA from other species, such as Escherichia coli, and could easily acquire a vancomycin-resistance gene from enterococci. Inactivation of this restriction system dramatically increases the transformation efficiency of clinical S. aureus strains, opening the field of molecular genetic manipulation of these strains using DNA of exogenous origin.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Transferência Genética Horizontal , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo I/antagonistas & inibidores , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Enterococcus faecalis/enzimologia , Enterococcus faecalis/genética , Escherichia coli/genética , Marcação de Genes , Genes Bacterianos , Humanos , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Dados de Sequência Molecular , Plasmídeos/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Staphylococcus aureus/isolamento & purificação
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