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1.
Am J Pathol ; 190(4): 862-873, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32200972

RESUMO

Group A streptococcus (GAS) is a major pathogen that impacts health and economic affairs worldwide. Although the oropharynx is the primary site of infection, GAS can colonize the female genital tract and cause severe diseases, such as puerperal sepsis, neonatal infections, and necrotizing myometritis. Our understanding of how GAS genes contribute to interaction with the primate female genital tract is limited by the lack of relevant animal models. Using two genome-wide transposon mutagenesis screens, we identified 69 GAS genes required for colonization of the primate vaginal mucosa in vivo and 96 genes required for infection of the uterine wall ex vivo. We discovered a common set of 39 genes important for GAS fitness in both environments. They include genes encoding transporters, surface proteins, transcriptional regulators, and metabolic pathways. Notably, the genes that encode the surface-exclusion protein (SpyAD) and the immunogenic secreted protein 2 (Isp2) were found to be crucial for GAS fitness in the female primate genital tract. Targeted gene deletion confirmed that isogenic mutant strains ΔspyAD and Δisp2 are significantly impaired in ability to colonize the primate genital tract and cause uterine wall pathologic findings. Our studies identified novel GAS genes that contribute to female reproductive tract interaction that warrant translational research investigation.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Doenças Vaginais/microbiologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica , Macaca fascicularis , Proteínas de Membrana/genética , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/metabolismo , Doenças Vaginais/patologia , Virulência
2.
Nat Commun ; 11(1): 742, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029734

RESUMO

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions.


Assuntos
Complexos Multiproteicos/química , Biblioteca de Peptídeos , Proteínas/química , Algoritmos , Benchmarking , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Reagentes para Ligações Cruzadas , Espectrometria de Massas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ferramenta de Busca/normas , Streptococcus pyogenes/química , Streptococcus pyogenes/genética
3.
J Med Microbiol ; 69(3): 443-450, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32011228

RESUMO

Introduction. Pharyngotonsillitis caused by Streptococcus pyogenes (group A streptococci, or GAS) is among the most common infections treated with antibiotics in pediatric patients.Aim. This study aimed to analyse changes in molecular epidemiology and antibiotic susceptibility among GAS isolates in three study periods spanning 10 years.Methodology. GAS isolated from paediatric patients with pharyngotonsillitis during Period I (mid-2007 to 2008, n=235), Period II (2012, n=210), and Period III (2018, n=189) were analysed for emm type, multilocus sequence type (MLST), antibiotic susceptibility, and macrolide (ML)- and quinolone (QL)-resistance genes.Results. Over 20 % of isolates represented emm1 and emm12 types, remaining common in all three periods. Among other emm types, emm4 was common in Period I, emm28 and emm89 in Period II, and emm3 and emm89 in Period III. All isolates remained highly susceptible to penicillins and cephalosporins. Isolates possessing mefA, ermA, or ermB genes mediating ML resistance increased from 34.9 % in Period I to 60.9 % in Period II, but fell to 27.5 % in Period III. QL-resistant isolates with amino acid substitutions affecting ParC and/or GyrA gradually increased from 11.5 to 14.3 %. Specific sequence types identified by MLST and emm typing were associated closely with ML or QL resistance.Conclusion. Our findings indicate that even in ambulatory care, antibiotic choice for these infections should be based on rapid identification and characterization of causative pathogens.


Assuntos
Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Farmacorresistência Bacteriana/genética , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/genética , Tonsilite/epidemiologia , Proteínas da Membrana Bacteriana Externa/genética , Técnicas de Tipagem Bacteriana , Criança , Pré-Escolar , Genótipo , Humanos , Japão/epidemiologia , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Nasofaringe/microbiologia , Filogenia , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/efeitos dos fármacos , Tonsilite/tratamento farmacológico , Tonsilite/microbiologia
4.
Nat Biomed Eng ; 4(1): 111-124, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31937939

RESUMO

The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from Streptococcus pyogenes (SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Aprendizado Profundo , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/fisiologia , Streptococcus pyogenes/genética
5.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31591169

RESUMO

As a strict human pathogen, Streptococcus pyogenes (group A Streptococcus, or GAS) causes a wide range of infections, from superficial to life-threatening diseases, upon dissemination. Thus, it is necessary to gain a better understanding of how GAS successfully overcomes host-mediated challenges and infects various host niches. We previously identified subcutaneous fitness (scf) genes in the clinically relevant wild-type (WT) GAS M1T1 5448 strain that are critical for fitness during murine soft-tissue infection at both 24 h and 48 h postinfection. The uncharacterized locus scfCDE was transcribed as an operon and is predicted to encode an ABC importer for nutrient uptake (e.g., amino acids). Individual scfCDE deletion mutants grew comparably to WT 5448 in rich medium but exhibited reduced fitness during competitive growth in murine soft tissue and in nutrient-limiting chemically defined medium (CDM). A deletion of the permease gene scfD resulted in a monoculture growth defect in CDM that could be rescued by addition of excess peptides, suggesting a role as an amino acid importer. Interestingly, the ΔscfC substrate-binding and ΔscfD permease mutants, but not the ΔscfE ATPase mutant, were highly attenuated in murine soft tissue. Moreover, all three genes were required for GAS survival in human blood, indicating their impact is not limited to superficial infections. As such, scfCDE plays an integral role in enhancing GAS adaptation during localized infection as well as dissemination to deeper host environments. Since scfCDE is conserved throughout Firmicutes, this work may contribute to the development of therapeutic strategies against GAS and other Gram-positive pathogens.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Regulação Bacteriana da Expressão Gênica , Camundongos , Infecções Estreptocócicas/genética , Virulência/genética
6.
BMC Infect Dis ; 19(1): 850, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615449

RESUMO

BACKGROUND: To determine, from October 2010 to October 2018, the epidemiology of Deep Neck Infections (DNIs), regarding the detection, the identification and the susceptibility to antimicrobials of causative microorganisms, in Thessaly-Central Greece. METHODS: An analysis of data from a prospective database was conducted on 610 consecutive patients with DNIs treated in the Otolaryngology / Head & Neck Surgery Department of University Hospital of Larissa. Demographics, clinical features and microbiological data were analyzed. RESULTS: Among the 610 patients (1,9/1 male to female ratio, mean age: 39,24 ± 17,25) with DNIs, 579 had a single space (94,9%), while the remaining 31 had a multi-space (5,1%) DNI. The most common areas affected were the peritonsillar space (84,6%) followed by the submandibular space (6,5%). Clinical samples were obtained from 462 patients, and were tested by culture and by the application of 16S rRNA PCR. Two hundred fifty-five samples (55,2%) gave positive cultures, in which Streptococcus pyogenes and Staphylococcus aureus were predominant. The application of the 16S rRNA PCR revealed that 183 samples (39,6%) were positive for bacterial DNA; 22 of them, culture negative, were found to be positive for anaerobic (Fusobacterium necrophorum, Actinomyces israellii etc) and for fastidious microorganisms (Brucella mellitensis, Mycobacterium avium). CONCLUSION: DNIs represent a medical and surgical emergency and evidence-guided empirical treatment with intravenous infusion of antibiotics at the time of diagnosis is mandatory, highlighting the importance of epidemiological studies regarding the causative microorganisms. Although, in our study, the predominant pathogens were S. pyogenes and S. aureus, the combination of culture and molecular assay revealed that anaerobic bacteria play also a significant role in the pathogenesis of DNIs. Based on the local epidemiology, we propose as empirical therapy the intravenous use of a beta-lactam /beta-lactamase inhibitor; metronidazole or clindamycin can be added only in specific cases such as in immunocompromised patients.


Assuntos
Anti-Infecciosos/farmacologia , Bacteriemia/diagnóstico , Bactérias/efeitos dos fármacos , Pescoço/microbiologia , Abscesso/diagnóstico , Abscesso/microbiologia , Adulto , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Bactérias/isolamento & purificação , Feminino , Grécia/epidemiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , RNA Ribossômico 16S/metabolismo , Estudos Retrospectivos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação
7.
PLoS Biol ; 17(10): e3000496, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31603896

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , RNA Guia/genética , Substituição de Aminoácidos , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Engenharia de Proteínas/métodos , RNA Guia/química , RNA Guia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
9.
J Med Microbiol ; 68(10): 1540-1543, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31483245

RESUMO

Four group A streptococcus (GAS) bacteraemia occurred in a small burn unit within 2 weeks. The GAS patient isolates, characterized as emm89, shared the same PFGE pulsotype with two other strains isolated 2 months later. The outbreak investigation revealed that a nurse was the most likely source of GAS transmission, as she was confirmed to carry the same outbreak strain in her throat and had direct and regular contact with the six outbreak patients in the unit. The outbreak was controlled after the nurse had undergone eradication treatment. This report highlights the emergence of the emm89 clone and its capacity to elicit invasive GAS outbreaks.


Assuntos
Unidades de Queimados/estatística & dados numéricos , Infecção Hospitalar/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/isolamento & purificação , Adulto , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Feminino , Humanos , Pessoa de Meia-Idade , Epidemiologia Molecular , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Tunísia , Adulto Jovem
10.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470538

RESUMO

Protein conjugations at post-translational levels are known to be essential to protein stability and function. Recently, it has been proven that the split protein CnaB2 (SpyTag/SpyCatcher, ST/SC) from Streptococcus pyogenes can induce covalent conjugation rapidly and efficiently under various conditions. The protein of interest fused with the split protein SC/ST could be assembled spontaneously. In light of this finding, we introduced the ST/SC protein coupling concept into the silkworm-bacmid protein expression system (SpyBEVS). As a proof of concept, we first examined and confirmed that a competent ligation occurred between ST/SC-fused protein partners in vitro in cultured silkworm cells and in vivo in silkworm larvae by co-infection of several recombinant baculoviruses. The protein conjugation could be also achieved sufficiently by a simple one-step mixture of purified ST/SC-tagged peptide-protein pairs in vitro. Given the flexibility and robustness of silkworm-BEVS, our results on SpyBEVS show an alternative method for enabling the production of protein decorations in vitro and inside of silkworms.


Assuntos
Bombyx/genética , Vetores Genéticos/genética , Proteínas de Insetos/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bombyx/citologia , Bombyx/metabolismo , Células Cultivadas , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Engenharia de Proteínas/métodos , Estabilidade Proteica , Reprodutibilidade dos Testes , Homologia de Sequência de Aminoácidos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
11.
Microbiol Immunol ; 63(10): 413-426, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31403217

RESUMO

Streptococcus pyogenes is a causative agent of streptococcal toxic shock syndrome (STSS). The complete genome sequence of a S. pyogenes strain 10-85 isolated from a STSS patient was recently announced. In this study, the genome sequence was dissected and it was found that the genomic region around 200 kbp (region A) and the genomic region around 1600 kbp (region B) were replaced by each other in strain 10-85, when compared with those in reference strains SF370 and A20. In order to address whether this replacement is unique to 10-85, we further analyzed 163 emm1-type strains. The results indicated that none of the strains isolated before 1990 had the replacement. In contrast, most of the strains isolated at least after 2000 appeared to have the 10-85-type replacement.


Assuntos
DNA Bacteriano/genética , Genoma Bacteriano/genética , Choque Séptico/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Sequência de Bases , Humanos , Prevalência , Streptococcus pyogenes/isolamento & purificação
12.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31373269

RESUMO

The group A streptococci are associated with a group of diseases affecting the heart, brain, and joints that are collectively referred to as acute rheumatic fever. The streptococcal immune-mediated sequelae, including acute rheumatic fever, are due to antibody and cellular immune responses that target antigens in the heart and brain as well as the group A streptococcal cross-reactive antigens as reviewed in this article. The pathogenesis of acute rheumatic fever, rheumatic heart disease, Sydenham chorea, and other autoimmune sequelae is related to autoantibodies that are characteristic of autoimmune diseases and result from the immune responses against group A streptococcal infection by the host. The sharing of host and streptococcal epitopes leads to molecular mimicry between the streptococcal and host antigens that are recognized by the autoantibodies during the host response. This article elaborates on the discoveries that led to a better understanding of the pathogenesis of disease and provides an overview of the history and the most current thought about the immune responses against the host and streptococcal cross-reactive antigens in group A streptococcal sequelae.


Assuntos
Antígenos de Bactérias/imunologia , Autoanticorpos/imunologia , Autoimunidade , Febre Reumática/imunologia , Streptococcus pyogenes/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Reações Cruzadas , Humanos , Mimetismo Molecular , Febre Reumática/microbiologia , Streptococcus pyogenes/química , Streptococcus pyogenes/genética
13.
J Hosp Infect ; 103(1): 21-26, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31283948

RESUMO

BACKGROUND: Whole genome sequencing (WGS) of Streptococcus pyogenes linked to invasive disease has been used to identify and investigate outbreaks. The clinical application of WGS in real-time for outbreak control is seldom employed. AIMS: A fatal case of bacteraemia at a national orthopaedic hospital prompted an outbreak investigation to identify carriers and halt transmission using real-time WGS. METHODS: Retrospective surveillance was conducted to identify patients with Streptococcus pyogenes infections in the last year. Upon contact tracing, four patients and 179 staff were screened for Streptococcus pyogenes carriage. All isolates identified were emm-typed. WGS was performed in real-time on a subset of isolates. FINDINGS: Twelve isolates of Streptococcus pyogenes from the index case, two patients and eight staff were identified. Six isolates were emm 1.0, including the index case and five staff isolates. The remaining isolates belonged to distinct emm types. WGS analysis was undertaken on the six emm 1.0 isolates. Five were indistinguishable by single nucleotide polymorphism (SNP) analysis, with 0 SNP distance, and one had one SNP difference, supporting the hypothesis of recent local transmission. All screen-positive healthcare workers were offered treatment with penicillin or clindamycin. No further cases were identified. CONCLUSION: The increased molecular discrimination of WGS confirmed the clustering of these cases and the outbreak was contained. This demonstrates the clinical utility of WGS in managing outbreaks of invasive Streptococcus pyogenes in real-time and we recommend its implementation as a routine clinical service.


Assuntos
Bacteriemia/epidemiologia , Portador Sadio/epidemiologia , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/classificação , Sequenciamento Completo do Genoma/métodos , Bacteriemia/microbiologia , Portador Sadio/microbiologia , Portador Sadio/transmissão , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , Transmissão de Doença Infecciosa/prevenção & controle , Hospitais , Humanos , Epidemiologia Molecular/métodos , Tipagem Molecular/métodos , Estudos Retrospectivos , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/transmissão , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação
14.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31267891

RESUMO

The human oral-nasal mucosa is the primary reservoir for Streptococcus pyogenes infections. Although the most common infection of consequence in temperate climates is pharyngitis, the past 25 years have witnessed a dramatic increase in invasive disease in many regions of the world. Historically, S. pyogenes has been associated with sepsis and fulminate systemic infections, but the mechanism by which these streptococci traverse mucosal or epidermal barriers is not understood. The discovery that S. pyogenes can be internalized by mammalian epithelial cells at high frequencies (1-3) and/or open tight junctions to pass between cells (4) provides potential explanations for changes in epidemiology and the ability of this species to breach such barriers. In this article, the invasins and pathways that S. pyogenes uses to reach the intracellular state are reviewed, and the relationship between intracellular invasion and human disease is discussed.


Assuntos
Receptores de Superfície Celular/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/fisiologia , Animais , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/microbiologia , Humanos , Faringite/genética , Faringite/metabolismo , Faringite/microbiologia , Receptores de Superfície Celular/genética , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/genética
15.
BMC Infect Dis ; 19(1): 633, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315580

RESUMO

BACKGROUND: Group A Streptococcal (GAS) infections cause the autoimmune disease acute rheumatic fever (ARF), which can progress to chronic rheumatic heart disease (RHD). Treating pharyngitis caused by GAS with antibiotics is important in preventing ARF. However, it is difficult to distinguish these infections from GAS carriers. There is growing evidence for GAS skin infections as a cause of ARF. This study will identify the incidence of true GAS pharyngitis and serological responses to GAS skin infections. The effectiveness of antibiotics for these conditions will be explored, and modifiable risk factors. Serum antibody titres indicating the upper limits of normal (ULN for ASO/ADB antibodies) will be established alongside carriage rates in asymptomatic children. METHODS: This is a prospective disease incidence study, with an associated case-control study. The study population includes 1000 children (5-14 years) from Auckland, New Zealand, 800 of whom have visited their healthcare professional, resulting in a throat or skin swab for GAS, and 200 who are asymptomatic. The conditions of interest are GAS throat swab positive pharyngitis (n = 200); GAS carriage (n = 200); GAS negative throat swab (n = 200); GAS skin infections (n = 200); and asymptomatic controls (n = 200). All participants, except asymptomatic controls, will have acute and convalescent serological testing for ASO/ADB titres (collected < 9 days, and 2-4 weeks following symptom onset, respectively), alongside viral PCR from throat swabs. Asymptomatic controls will have ASO/ADB titres measured in one blood specimen and a throat swab for microbial culture. Caregivers of children will be interviewed using a questionnaire and any GAS isolates identified will be emm typed. The persistence of GAS antibodies will also be investigated. DISCUSSION: Findings from this study will fill critical gaps in scientific knowledge to better understand the pathophysiology of ARF, improve clinical management of GAS infections, and design more effective ARF prevention programmes. In particular it will measure the incidence of true, serologically confirmed GAS pharyngitis; assess the immune response to GAS skin infections and its role as a cause of ARF; examine the effectiveness of oral antibiotics for treating GAS pharyngitis and carriage; and identify whether risk factors for GAS infections might provide intervention points for reducing ARF.


Assuntos
Faringite/microbiologia , Febre Reumática/microbiologia , Dermatopatias Bacterianas/microbiologia , Adolescente , Antibacterianos/uso terapêutico , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Incidência , Masculino , Nova Zelândia/epidemiologia , Faringite/tratamento farmacológico , Faringite/epidemiologia , Reação em Cadeia da Polimerase , Estudos Prospectivos , Febre Reumática/tratamento farmacológico , Febre Reumática/epidemiologia , Dermatopatias Bacterianas/tratamento farmacológico , Dermatopatias Bacterianas/epidemiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação , Streptococcus pyogenes/patogenicidade
17.
J Med Microbiol ; 68(7): 1053-1058, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31169483

RESUMO

BACKGROUND: Streptococcus pyogenes is the most common cause of bacterial pharyngitis. Genotyping of emm is useful for molecular epidemiological survey of S. pyogenes. Antibiotic resistance data are needed for empirical treatments. METHODS: In total, 358 children in Changwon, Korea who had pharyngitis symptoms were subjected to throat cultures to isolate S. pyogenes in 2017. emm genotyping was performed by direct sequencing. An antibiotic susceptibility test was performed using the disk diffusion method for erythromycin (ERY), clindamycin (CLI), tetracycline (TET) and ofloxacin (OFX). Screening for macrolide resistance phenotype and its determinants was performed for the ERY-resistant strains. RESULTS: A total of 190 strains (53.1 %) of S. pyogenes were isolated from 358 children. The most frequent emm genotype was emm4 (53.2 %), followed by emm89 (12.6 %), emm28 (11.6 %) and emm1 (10 %). Antibiotic resistance rates to ERY, CLI, TET and OFX were 3.2 %, 2.6 %, 1.1 % and 2.6%, respectively. There were five isolates of the cMLSB phenotype having the ermB gene and one M phenotype harbouring the mefA gene. CONCLUSIONS: The distribution of emm genotypes was quite different from those previously reported in Korea. emm4 accounted for more than 50  % of the genotypes. Macrolide resistance rates remained very low, but five of six ERY-resistant strains displayed the cMLSB phenotype.


Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Farmacorresistência Bacteriana/genética , Faringite/epidemiologia , Faringite/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Adolescente , Antibacterianos/farmacologia , Criança , Pré-Escolar , Genótipo , Humanos , República da Coreia/epidemiologia , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/efeitos dos fármacos
18.
J Med Microbiol ; 68(7): 1059-1071, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31192782

RESUMO

PURPOSE: Unlike western countries the knowledge of group A streptococcus (GAS) epidemiology in India remains patchy and incomplete. Typing is crucial for surveillance as well as in predicting the efficacy of multivalent M protein vaccine. The present study aimed to explore the emm types of 206 invasive and non-invasive GAS isolates from South India as well as reviewing all the published literature on GAS molecular epidemiology from India thereby generating a pan-Indian data to predict the conjectural coverage of the 30-valent M-protein vaccine in this population. METHODOLOGY: emm typing and superantigen (SAg) profiling of GAS along with reviewing literatures on GAS molecular epidemiology from India. RESULTS: This study revealed a high diversity of emm types with emm 63, 82, 183, 85, 92, 169, 42, 44, 106, 74, 12 being frequently encountered, belonging to twenty emm clusters. The pan-Indian data on prevalent emm types further supports our study findings with 135 emm different types. Six clusters dominated accounting for 80 % of the GAS isolates: E3(26 %), E6(20 %), E2(11 %), E4(10 %), D4(7 %), E1(6 %). No significant association was noted between emm types and the nature of infection (P≥0.05) while a few SAg profiles were significantly associated with certain emm types. Pan Indian data revealed that only 16 % of the emm types encountered were included in proposed 30-valent M protein based vaccine. CONCLUSION: The coverage among the South Indian GAS isolates was 28.2 % which increased to only 46.6 % with the cross-opsonic effect, thus highlighting the importance of developing a specific multivalent vaccine including the prevalent emm types in India or considering the use of conserved C-repeat vaccines and non-M protein based vaccines.


Assuntos
Epidemiologia Molecular , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Adolescente , Adulto , Proteínas de Bactérias/genética , Criança , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/imunologia , Adulto Jovem
19.
Plant Cell Physiol ; 60(10): 2255-2262, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31198958

RESUMO

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), comprising an RNA-guided DNA endonuclease and a programmable guide RNA (gRNA), is currently recognized to be a powerful genome-editing tool and is widely used in biological science. Despite the usefulness of the system, a protospacer-adjacent motif (PAM) immediately downstream of the target sequence needs to be taken into account in the design of the gRNA, a requirement which limits the flexibility of the CRISPR-based genome-editing system. To overcome this limitation, a Cas9 isolated from Streptococcus pyogenes, namely SpCas9, engineered to develop several variants of Cas9 nuclease, has been generated. SpCas9 recognizes the NGG sequence as the PAM, whereas its variants are capable of interacting with different PAMs. Despite the potential advantage of the Cas9 variants, their functionalities have not previously been tested in the widely used model plant, Arabidopsis thaliana. Here, we developed a plant-specific vector series harboring SpCas9-VQR (NGAN or NGNG) or SpCas9-EQR (NGAG) and evaluated their functionalities. These modified Cas9 nucleases efficiently introduced mutations into the CLV3 and AS1 target genes using gRNAs that were compatible with atypical PAMs. Furthermore, the generated mutations were passed on to their offspring. This study illustrated the usefulness of the SpCas9 variants because the ability to generate heritable mutations will be of great benefit in molecular genetic analyses. A greater number of potential SpCas9-variant-recognition sites in these genes are predicted, compared with those of conventional SpCas9. These results demonstrated the usefulness of the SpCas9 variants for genome editing in the field of plant science research.


Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , RNA Guia/genética , Streptococcus pyogenes/enzimologia , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Mutagênese , Mutação , Especificidade da Espécie , Streptococcus pyogenes/genética
20.
PLoS Pathog ; 15(6): e1007841, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206562

RESUMO

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (ΔRSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ΔRSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ΔRSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ΔRSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Metilação de DNA , Enzimas de Restrição-Modificação do DNA , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica , Streptococcus pyogenes , Animais , Proteínas de Bactérias/genética , Citocinas/metabolismo , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Fasciite Necrosante/genética , Fasciite Necrosante/metabolismo , Fasciite Necrosante/patologia , Feminino , Humanos , Camundongos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade
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