RESUMO
The obligate intracellular human pathogenic bacterium Chlamydia trachomatis has evolved multiple mechanisms to circumvent the host immune system. Infected cells exhibit a profound resistance to the induction of apoptosis and down-regulate the expression of major histocompatibility complex class I and class II molecules to evade the cytotoxic effect of effector immune cells. Here we demonstrate the down-regulation of tumor necrosis factor receptor 1 (TNFR1) on the surface of infected cells. Interestingly, other members of the TNFR family such as TNFR2 and CD95 (Fas/Apo-1) were not modulated during infection, suggesting a selective mechanism underlying surface reduction of TNFR1. The observed effect was not due to reduced expression since the overall amount of TNFR1 protein was increased in infected cells. TNFR1 accumulated at the chlamydial inclusion and was shed by the infected cell into the culture supernatant. Receptor shedding depended on the infection-induced activation of the MEK-ERK pathway and the metalloproteinase TACE (TNFalpha converting enzyme). Our results point to a new function of TNFR1 modulation by C. trachomatis in controlling inflammatory signals during infection.
Assuntos
Proteínas ADAM/imunologia , Apoptose/imunologia , Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Proteínas ADAM/metabolismo , Proteína ADAM17 , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/microbiologia , Células Jurkat , MAP Quinase Quinase Quinases/imunologia , MAP Quinase Quinase Quinases/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Células U937 , Receptor fas/imunologia , Receptor fas/metabolismoRESUMO
Anaemia of chronic disease (ACD) is a common finding involving iron deficiency and signs of inflammation. Here, we report on two patients with ACD where a persistent infection with Chlamydophila (Chlamydia) pneumoniae (CP) was detected in bone marrow (BM) biopsies. Infection was suspected by routine cytology and confirmed by immunofluorescence, electron microscopy, polymerase chain reaction (PCR) including different primer sets and laboratories and sequencing of the PCR product. This is a first report of chlamydial presence in the BM of anaemic patients. The cases are presented because persistent chlamydial infection may contribute more frequently to chronic refractory anaemia than previously suspected.
Assuntos
Anemia/etiologia , Anemia/microbiologia , Medula Óssea/microbiologia , Infecções por Chlamydia/complicações , Chlamydophila pneumoniae/isolamento & purificação , Chlamydophila pneumoniae/patogenicidade , Adulto , Sequência de Bases , Medula Óssea/ultraestrutura , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/microbiologia , Chlamydophila pneumoniae/genética , Doença Crônica , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Microscopia EletrônicaRESUMO
Polymorphic membrane protein (Pmp)21 otherwise known as PmpD is the longest of 21 Pmps expressed by Chlamydophila pneumoniae. Recent bioinformatical analyses annotated PmpD as belonging to a family of exported Gram-negative bacterial proteins designated autotransporters. This prediction, however, was never experimentally supported, nor was the function of PmpD known. Here, using 1D and 2D PAGE we demonstrate that PmpD is processed into two parts, N-terminal (N-pmpD), middle (M-pmpD) and presumably third, C-terminal part (C-pmpD). Based on localization of the external part on the outer membrane as shown by immunofluorescence, immuno-electron microscopy and immunoblotting combined with trypsinization, we demonstrate that N-pmpD translocates to the surface of bacteria where it non-covalently binds other components of the outer membrane. We propose that N-pmpD functions as an adhesin, as antibodies raised against N-pmpD blocked chlamydial infectivity in the epithelial cells. In addition, recombinant N-pmpD activated human monocytes in vitro by upregulating their metabolic activity and by stimulating IL-8 release in a dose-dependent manner. These results demonstrate that N-PmpD is an autotransporter component of chlamydial outer membrane, important for bacterial invasion and host inflammation.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Chlamydia/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Membrana Celular/ultraestrutura , Chlamydia/isolamento & purificação , Chlamydia/metabolismo , Chlamydia/ultraestrutura , Eletroforese em Gel Bidimensional , Humanos , Neoplasias Laríngeas , Dados de Sequência Molecular , Monócitos/microbiologia , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Tripsina , Células Tumorais CultivadasRESUMO
Chlamydophila pneumoniae is an obligate intracellular pathogen implicated in a variety of acute and chronic diseases. Long-term infections are associated with a persistent life stage, in which bacteria can stay for years. They are less accessible to antibiotic treatment but still prone to sustain an inflammatory response. Different in vitro models have been established to mimic and characterize chlamydial persistency. For C. pneumoniae and Chlamydia trachomatis, altered metabolic activities and changed antigenic profiles compared to acute infections have been reported. Most studies including transcriptome and proteome analyses describe persistency induced by IFNgamma treatment. Here, we use iron depletion of the infected cell culture that also leads into persistent infection. We describe differently regulated proteins found by subtractive proteome analysis comparing two early stages of infection with and without addition of the iron chelator deferoxamine-mesylate. While only one bacterial protein was up-regulated during iron deficiency up to 24 h post infection (p.i.), 11 were found to be up-regulated and eight to be down-regulated from 24-48 h p.i. Two down-regulated proteins could be identified by peptide mass fingerprinting as thioredoxin reductase and chromosome partitioning protein (ParB). The latter is involved in chromosome segregation. Thus, using a comparative approach we identified on a proteome level down-regulation of ParB in persistent chlamydial forms, which is in agreement with previous results describing changes in cell division and atypical altered morphology of persistent Chlamydiae.
Assuntos
Infecções por Chlamydophila/metabolismo , Chlamydophila pneumoniae/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Deficiências de Ferro , Proteoma , Sequência de Aminoácidos , Antígenos de Bactérias , Autorradiografia , Fenômenos Fisiológicos Bacterianos , Divisão Celular , Linhagem Celular , Infecções por Chlamydophila/genética , Regulação para Baixo , Eletroforese em Gel Bidimensional , Epitélio/microbiologia , Corantes Fluorescentes/farmacologia , Humanos , Interferon gama/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Tripsina/farmacologia , Regulação para CimaRESUMO
Chlamydial protease-like activity factor (CPAF) is secreted to the cytoplasm of the infected cells where it proteolytically cleaves eukaryotic transcription factor RFX5. Here, we determined the localization pattern of CPAF during the course of an acute and persistent in vitro infection of the epithelial cell line HEp-2 with Chlamydophila pneumoniae strain VR1310. Using immunoblotting, confocal microscopy and electron microscopy, we found CPAF in the inclusion lumen or associated with bacteria during the first 48 h of an acute infection. Seventy-two hours and later, CPAF was present predominantly in the cytoplasm of the infected cells. Translocation of CPAF into cytoplasm correlated in time with degradation of the transcription factor RFX5, as confirmed by immunoblotting. Interestingly, during the persistent infection induced by either IFN-gamma or iron limitation CPAF translocation to the cytoplasm was inhibited resulting in unaffected or only partially reduced levels of RFX5. Based on presented findings, we propose that CPAF translocation to the cytoplasm is separated from its production. The translocation mechanism appears to be fully active during an acute infection; however, it is fully or partially inhibited during persistent infection induced by IFN-gamma or by iron limitation respectively. Consequently, this work demonstrates the importance of subcellular localization of CPAF for the characteristics of chlamydial acute and persistent infection in epithelial HEp-2 cells.