RESUMO
The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient's antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient's serum. Finally, we characterize one strain that was poorly recognized by the patient's antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.
Assuntos
Adaptação Fisiológica , Bordetella/fisiologia , Imunidade Adaptativa , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Bordetella/genética , Bordetella/imunologia , Feminino , Humanos , Evasão da Resposta Imune , Imunização , Pneumopatias/sangue , Pneumopatias/imunologia , Pneumopatias/microbiologia , Camundongos , Pessoa de Meia-Idade , Mutação , Antígenos O/genética , Análise de SequênciaRESUMO
BACKGROUND: While Staphylococcus epidermidis is commonly isolated from healthy human skin, it is also the most frequent cause of nosocomial infections on indwelling medical devices. Despite its importance, few genome sequences existed and the most frequent hospital-associated lineage, ST2, had not been fully sequenced. RESULTS: We cultivated 71 commensal S. epidermidis isolates from 15 skin sites and compared them with 28 nosocomial isolates from venous catheters and blood cultures. We produced 21 commensal and 9 nosocomial draft genomes, and annotated and compared their gene content, phylogenetic relatedness and biochemical functions. The commensal strains had an open pan-genome with 80% core genes and 20% variable genes. The variable genome was characterized by an overabundance of transposable elements, transcription factors and transporters. Biochemical diversity, as assayed by antibiotic resistance and in vitro biofilm formation, demonstrated the varied phenotypic consequences of this genomic diversity. The nosocomial isolates exhibited both large-scale rearrangements and single-nucleotide variation. We showed that S. epidermidis genomes separate into two phylogenetic groups, one consisting only of commensals. The formate dehydrogenase gene, present only in commensals, is a discriminatory marker between the two groups. CONCLUSIONS: Commensal skin S. epidermidis have an open pan-genome and show considerable diversity between isolates, even when derived from a single individual or body site. For ST2, the most common nosocomial lineage, we detect variation between three independent isolates sequenced. Finally, phylogenetic analyses revealed a previously unrecognized group of S. epidermidis strains characterized by reduced virulence and formate dehydrogenase, which we propose as a clinical molecular marker.
Assuntos
Infecções Relacionadas a Cateter/microbiologia , Infecção Hospitalar/microbiologia , Análise de Sequência de DNA/métodos , Pele/microbiologia , Staphylococcus epidermidis/classificação , Staphylococcus epidermidis/genética , Farmacorresistência Bacteriana , Evolução Molecular , Variação Genética , Genoma Bacteriano , Humanos , Dados de Sequência Molecular , Tipagem Molecular , Filogenia , Staphylococcus epidermidis/isolamento & purificaçãoRESUMO
The opportunistic human pathogen Pseudomonas aeruginosa causes rapidly progressive and tissue-destructive infections, such as hospital-acquired and ventilator-associated pneumonias. Innate immune responses are critical in controlling P. aeruginosa in the mammalian lung, as demonstrated by the increased susceptibility of MyD88(-/-) mice to this pathogen. Experiments conducted using bone marrow chimeric mice demonstrated that radio-resistant cells participated in initiating MyD88-dependent innate immune responses to P. aeruginosa. In this study we used a novel transgenic mouse model to demonstrate that MyD88 expression by epithelial cells is sufficient to generate a rapid and protective innate immune response following intranasal infection with P. aeruginosa. MyD88 functions as an adaptor for many TLRs. However, mice in which multiple TLR pathways (e.g., TLR2/TLR4/TLR5) are blocked are not as compromised in their response to P. aeruginosa as mice lacking MyD88. We demonstrate that IL-1R signaling is an essential element of MyD88-dependent epithelial cell responses to P. aeruginosa infection.
Assuntos
Fator 88 de Diferenciação Mieloide/imunologia , Mucosa Respiratória/microbiologia , Transdução de Sinais/imunologia , Animais , Humanos , Imunidade Inata , Interleucina-1/metabolismo , Camundongos , Camundongos Knockout , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/imunologiaRESUMO
Microbe-associated molecular patterns are recognized by Toll-like receptors of the innate immune system. This recognition enables a rapid response to potential pathogens but does not clearly provide a way for the innate immune system to discriminate between virulent and avirulent microbes. We find that pulmonary infection of mice with type 3 translocation-competent Pseudomonas aeruginosa triggers a rapid inflammatory response, while infection with isogenic translocation-deficient mutants does not. Discrimination between translocon-positive and -negative bacteria requires caspase-1 activity in bone marrow-derived cells and interleukin-1 receptor signaling. Thus, the activation of caspase-1 by bacteria expressing type 3 secretion systems allows for rapid recognition of bacteria expressing conserved functions associated with virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Caspase 1/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/imunologia , Células da Medula Óssea/imunologia , Células Epiteliais/microbiologia , Imunidade Inata , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Traqueia/citologia , Traqueia/microbiologia , VirulênciaRESUMO
Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections in immunocompromised individuals. P. aeruginosa employs a type III secretion system to inject effector molecules into the cytoplasm of the host cell. This interaction with the host cell leads to inflammatory responses that eventually result in cell death. We show that infection of macrophages with P. aeruginosa results in activation of caspase-1 in an IPAF-dependent, but flagellin-independent, manner. Macrophages deficient in IPAF or caspase-1 were markedly resistant to P. aeruginosa-induced cell death and release of the proinflammatory cytokine interleukin (IL)-1beta. A subset of P. aeruginosa isolates express the effector molecule exoenzyme U (ExoU), which we demonstrate is capable of inhibiting caspase-1-driven proinflammatory cytokine production. This study shows a key role for IPAF and capase-1 in innate immune responses to the pathogen P. aeruginosa, and also demonstrates that virulent ExoU-expressing strains of P. aeruginosa can circumvent this innate immune response.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/metabolismo , Animais , Caspase 1/metabolismo , Morte Celular , Citoplasma/metabolismo , Ativação Enzimática , Flagelina/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Cinética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos BiológicosRESUMO
XLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-gamma and decreased T(H)2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca(2+) mobilization and ERK activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation, and nuclear NF-kappaB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-theta recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-theta/NF-kappaB1 activation and suggesting a role for this pathway in T(H)2 regulation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Isoenzimas/fisiologia , NF-kappa B/metabolismo , Proteína Quinase C/fisiologia , Células Th2/citologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Antígenos CD , Proteína 10 de Linfoma CCL de Células B , Diferenciação Celular , Citocinas/biossíntese , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA3 , Glicoproteínas/fisiologia , Imunoglobulinas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C-theta , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-fyn , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Superfície Celular , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Transativadores/genéticaRESUMO
2B4 (CD244) is a receptor belonging to the CD2-signaling lymphocytic activation molecule family and is found on all murine NK cells and a subset of NKT and CD8+ T cells. Murine 2B4 is expressed as two isoforms (2B4 short and 2B4 long) that arise by alternative splicing. They differ only in their cytoplasmic domains and exhibit opposing function when expressed in the RNK-16 cell line. The ligand for 2B4, CD48, is expressed on all hemopoietic cells. Previous studies have shown that treatment of NK cells with a 2B4 mAb results in increased cytotoxicity and IFN-gamma production. In this report, we used CD48+/- variants of the P815 tumor cell line and 2B4 knockout mice to show that engagement of 2B4 by its counterreceptor, CD48, expressed on target cells leads to an inhibition in NK cytotoxicity. The addition of 2B4 or CD48 mAb relieves this inhibition resulting in enhanced target cell lysis. This 2B4-mediated inhibition acts independently of signaling lymphocytic activation molecule-associated protein expression. Imaging studies show that 2B4 preferentially accumulates at the interface between NK and target cells during nonlytic events also indicative of an inhibitory receptor. This predominant inhibitory function of murine 2B4 correlates with increased 2B4 long isoform level expression over 2B4 short.