RESUMEN
During persistent infection, the intracellular bacterial pathogen Chlamydia trachomatis is viable but severely attenuates the production of new, infectious elementary bodies (EBs). To investigate the reasons for this lack of new EB output, we analysed the expression of chlamydial genes encoding products required for DNA replication and cell division, using in vitro models of active versus persistent infection and synovial tissue samples from patients with chronic Chlamydia-associated arthritis. Hep-2 cells were infected with K serovar C. trachomatis and harvested at t = 0-48 h post-infection (p.i; active). Human monocytes were infected similarly and harvested at t = 1-7 days p.i. (persistent). RNA preparations from infected/uninfected cells and patient samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) targeting polA, dnaA, mutS and parB mRNA, related to chlamydial DNA replication/segregation; these were expressed in infected Hep-2 cells from 11 to 48 h p.i; ftsK and ftsW, related to cell division, were expressed similarly. Real-time PCR analyses demonstrated that significant accumulation of chlamydial chromosome began at about 12 h p.i. in infected Hep-2 cells. In infected human monocytes, polA, dnaA, mutS and parB mRNA were produced from days 1-7 p.i. and were weakly expressed in patient samples. Real-time PCR indicated the continuing accumulation of chlamydial chromosome during the 7 day monocyte infection, although the rate of such accumulation was lower than that occurring during active growth. However, transcripts from ftsK and ftsW were detected only at 1 day p.i. in infected monocytes but not thereafter, and they were absent in all patient samples. Thus, genes whose products are required for chlamydial DNA replication are expressed during persistence, but transcription of genes whose products are required for cytokinesis is severely downregulated. These data explain, at least in part, the observed attenuation of new EB production during chlamydial persistence.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chlamydia trachomatis/genética , Replicación del ADN/genética , Genes Bacterianos/genética , Monocitos/microbiología , Proteínas Bacterianas/genética , División Celular/genética , Chlamydia trachomatis/citología , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/metabolismo , Cromosomas Bacterianos/genética , Cartilla de ADN , Regulación Bacteriana de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Modelos Biológicos , ARN Bacteriano/análisis , ARN Bacteriano/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Líquido Sinovial/microbiología , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Viruses can escape T-cell surveillance by infecting macrophages and thereby induce apoptosis of noninfected T cells. This ability had not been demonstrated for bacteria. We investigated whether infection of macrophages with the important human pathogen Chlamydia trachomatis can induce T-cell apoptosis. Because Chlamydia-Mycoplasma coinfection is a frequent event, the ability of Mycoplasma fermentans-infected macrophages to induce T-cell apoptosis was also studied. Infected macrophages were cocultivated with autologous T cells in different activation states. Propidium iodide-based fluorescence-activated cell sorter analysis demonstrated that macrophages infected with viable chlamydiae induced T-cell death. Apoptosis was identified as the mode of death induction by using a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay. Induction of T-cell death was macrophage dependent. Incubation of T cells with infectious chlamydiae in the absence of macrophages did not lead to T-cell apoptosis. UV irradiation of chlamydiae diminished the ability to induce death. T-cell death was induced by a cell-free supernatant of infected macrophages. Not only phytohemagglutinin-preactivated T cells but also non-mitogen-preactivated T cells were susceptible to C. trachomatis-induced apoptosis. In contrast, M. fermentans infection of macrophages did not induce T-cell death. Coinfection had no additional effect. In summary, intracellular chlamydial infection of macrophages can induce T-cell apoptosis. Apoptosis induction by chlamydiae possibly explains how persistently infected macrophages escape T-cell surveillance and why the Chlamydia-specific T-cell response is diminished during persistent chlamydial infection.
Asunto(s)
Apoptosis , Chlamydia trachomatis/patogenicidad , Macrófagos/microbiología , Linfocitos T/fisiología , Chlamydia trachomatis/efectos de la radiación , Humanos , Activación de Linfocitos , Macrófagos/fisiología , Fitohemaglutininas/farmacología , Rayos UltravioletaRESUMEN
Several strains of Chlamydia trachomatis (CT) and C. pneumoniae (CP) from different sources were screened for mycoplasma contamination using a sensitive nested 16S rDNA polymerase chain reaction-specific for a broad range of mycoplasma species. Five of nine CT and 5/16 CP isolates were contaminated by mycoplasma. Mycoplasma fermentans, M. hyorhinis and M. hominis were found as contaminating agents. To our knowledge no data are available on whether coinfection of chlamydia with mycoplasma alters the biological behavior of chlamydia. Analysis of the biological effect of mycoplasma on chlamydial infection showed a profound mycoplasma-induced reduction of chlamydial growth. Mycoplasma were efficiently eliminated from chlamydial cultures in HEp-2 cells by treatment with mupirocin without affecting chlamydial replication or host cell growth. Two chlamydial strains, C. trachomatis serovar K and one clinical isolate of C. pneumoniae were purged by this method.