RESUMO
Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall-anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28Nt, R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28Nt and both subdomains interacted directly with the laminin-binding α3ß1, α6ß1, and α6ß4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28Nt also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Adesão Celular/fisiologia , Integrina alfa3beta1/metabolismo , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/metabolismo , Adesinas Bacterianas/química , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/química , Linhagem Celular Tumoral , Endométrio/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Queratinócitos/metabolismo , Ligação Proteica , Domínios Proteicos , Streptococcus pyogenes/química , Células Estromais/metabolismoRESUMO
An outbreak of nosocomial infections due to Streptococcus pyogenes (Group A Streptococcus; GAS) occurred in a post-surgery oncology unit and concerned more than 60 patients and lasted 20 months despite enhanced infection control and prophylaxis measures. All GAS strains were characterized (emm genotype, toxin gene profile and pulse-field gel electrophoresis subtype). Selected strains were sequenced and phylogenetic relationship established. Capacity to form biofilm and interaction with human pulmonary epithelial cells and macrophages were determined. Twenty-six GAS strains responsible for invasive infections (II) and 57 for non-II or colonization were isolated from patients (n = 66) or healthcare workers (n = 13). Seventy strains shared the same molecular markers and 69 the same PFGE pattern; 56 were sequenced. They all belonged to the emerging emm89 clade 3; all but 1 were clonal. Whole genome sequencing identified 43 genetic profiles with sporadic mutations in regulatory genes and acquired mutations in 2 structural genes. Except for two regulatory gene mutants, all strains tested had the same biofilm formation capacity and displayed similar adherence and invasion of pulmonary epithelial cells and phagocytosis and survival in human macrophages. This large outbreak of GAS infection in a post-surgery oncology unit, a setting that contains highly susceptible patients, arose from a strain of the emergent emm89 clade. No relationship between punctual or acquired mutations, invasive status, and strain phenotypic characteristics was found. Noteworthy, the phenotypic characteristics of this clone account for its emergence and its remarkable capacity to elicit outbreaks.
Assuntos
Surtos de Doenças , Genótipo , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/isolamento & purificação , Infecção da Ferida Cirúrgica/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Toxinas Bacterianas/análise , Biofilmes/crescimento & desenvolvimento , Eletroforese em Gel de Campo Pulsado , Células Epiteliais/microbiologia , Feminino , França , Técnicas de Genotipagem , Humanos , Macrófagos/microbiologia , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Neoplasias/cirurgia , Filogenia , Análise de Sequência de DNA , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/crescimento & desenvolvimento , Infecção da Ferida Cirúrgica/microbiologia , Adulto JovemRESUMO
Group A Streptococcus (GAS) infections present high morbidity and mortality rates and consequently remain a significant health problem. The emm3 isolates induce more severe pathologies than all others. In this study, we tested, on a collection of invasive and non-invasive emm3 clinical isolates, whether in that genotype the invasive status of the strains affects the innate immune response. We show that phagocytosis is dependent on the invasiveness of the isolates. Interestingly, all emm3 isolates compromise macrophage integrity, already noticeable 1 h after infection. Inflammatory modulators (IL-6, TNF-α and IFN-ß) are nevertheless detected during at least 6 h post-infection. This is a likely consequence of the macrophages not being all infected. The efficient and rapid induction of macrophage death could explain the virulence of the emm3 strains.
Assuntos
Macrófagos/imunologia , Macrófagos/microbiologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/fisiologia , Animais , Caspase 3/metabolismo , Morte Celular/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Variação Genética , Genótipo , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Fagocitose/imunologia , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/classificação , Streptococcus pyogenes/isolamento & purificação , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
Group A Streptococcus (GAS) infections remain a significant health care problem due to high morbidity and mortality associated with GAS diseases, along with their increasing worldwide prevalence. Macrophages play a key role in the control and clearance of GAS infections. Moreover, pro-inflammatory cytokines production and GAS persistence and invasion are related. In this study we investigated the correlation between the GAS clinical isolates genotypes, their known clinical history, and their ability to modulate innate immune response. We constituted a collection of 40 independent GAS isolates representative of the emm types currently prevalent in France and responsible for invasive (57.5%) and non-invasive (42.5%) clinical manifestations. We tested phagocytosis and survival in mouse bone marrow-derived macrophages and quantified the pro-inflammatory mediators (IL-6, TNF-α) and type I interferon (INF-ß) production. Invasive emm89 isolates were more phagocytosed than their non-invasive counterparts, and emm89 isolates more than the other isolates. Regarding the survival, differences were observed depending on the isolate emm type, but not between invasive and non-invasive isolates within the same emm type. The level of inflammatory mediators produced was also emm type-dependent and mostly invasiveness status independent. Isolates of the emm1 type were able to induce the highest levels of both pro-inflammatory cytokines, whereas emm89 isolates induced the earliest production of IFN-ß. Finally, even within emm types, there was a variability of the innate immune responses induced, but survival and inflammatory mediator production were not linked.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Fasciite Necrosante/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Células da Medula Óssea/citologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fasciite Necrosante/microbiologia , Fasciite Necrosante/patologia , Genótipo , Humanos , Imunidade Inata , Interferon beta/análise , Interferon beta/metabolismo , Interleucina-6/análise , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Fagocitose , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/isolamento & purificação , Fator de Necrose Tumoral alfa/análiseRESUMO
Linezolid has emerged as an important therapeutic option for the treatment of Staphylococcus aureus in patients with cystic fibrosis. We report the rapid emergence, upon treatment with linezolid, of linezolid-resistant S. aureus clinical isolates through the accumulation of resistance-associated 23S rRNA mutations, together with acquisition of an altered mutator phenotype.
Assuntos
Antibacterianos/farmacologia , Fibrose Cística/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Acetamidas , Adulto , Antibacterianos/uso terapêutico , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Humanos , Linezolida , Oxazolidinonas , RNA Ribossômico 23S/genética , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidadeRESUMO
The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.