RESUMEN
Chlamydia trachomatis is a bacterial obligate intracellular parasite that replicates within a vacuole, termed an inclusion, that does not fuse with lysosomes. Within 2 h after internalization, the C. trachomatis inclusion ceases to interact with the endocytic pathway and, instead, becomes fusogenic with exocytic vesicles containing exogenously synthesized NBD-sphingomyelin. Both fusion of exocytic vesicles and long-term avoidance of lysosomal fusion require early chlamydial gene expression. Modification of the chlamydial inclusion probably occurs through the expression and insertion of chlamydial protein(s) into the inclusion membrane. To identify candidate inclusion membrane proteins, antisera were raised against a total membrane fraction purified from C. trachomatis-infected HeLa cells. By indirect immunofluorescence, this antisera recognized the inclusion membrane and, by immunoblot analysis, recognized three chlamydial-specific antigens of approximate molecular weights 15, 18 and 21 kDa. IncG, encoding an 18 kDa and 21 kDa doublet chlamydial antigen, was identified by screening a C. trachomatis, serovar L2, genomic expression library. Three additional genes, incD, incE and incF, were co-transcribed with incG. Monospecific antisera against each of the four genes of this operon demonstrated that the gene products were localized to the chlamydial inclusion membrane. Immediately downstream from the operon containing incD-G was the C. trachomatis homologue of incA. Like IncD, E, F and G, C. trachomatis IncA is also localized to the inclusion membrane. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that IncD-G, but not incA, are transcribed within the first 2 h after internalization, making them candidates for chlamydial factors required for the modification of the nascent chlamydial inclusion.
Asunto(s)
Chlamydia trachomatis/genética , Proteínas de la Membrana/genética , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Infecciones por Chlamydia/genética , Chlamydia trachomatis/patogenicidad , Endocitosis , Técnica del Anticuerpo Fluorescente , Regulación Bacteriana de la Expresión Génica , Células HeLa , Humanos , Proteínas de la Membrana/inmunología , Microscopía Inmunoelectrónica , Operón , Mapeo Físico de Cromosoma , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacuolas/microbiologíaRESUMEN
Following biosynthesis, class II MHC molecules are transported through a lysosome-like compartment, where they acquire antigenic peptides for presentation to T cells at the cell surface. This compartment is characterized by the presence of HLA-DM, which catalyzes the peptide loading process. Here we report that the morphology and function of the class II loading compartment is affected in diseases with a phenotypic change in lysosome morphology. Swollen lysosomes are observed in cells from patients with the hereditary immunodeficiency Chediak-Higashi syndrome and in cells infected with Coxiella burnetii, the rickettsial organism that causes Q fever. In both disease states, we observed that HLA-DR and HLA-DM accumulate in enlarged intracellular compartments, which label with the lysosomal marker LAMP-1. The distribution of class I MHC molecules was not affected, localizing disease effects to the endocytic pathway. Thus, cellular mechanisms controlling lysosome biogenesis also affect formation of the class II loading compartment. Analysis of cell surface class II molecules revealed that their steady-state levels were not reduced on diseased cells. However, in both disease states, enhanced interaction between HLA-DR and HLA-DM was detected. In the Chediak-Higashi syndrome cells, this correlated with more efficient removal of the CLIP peptide. These findings suggest a mechanism for perturbation of Ag presentation by class II molecules and consequent immune deficiencies in both diseases.
Asunto(s)
Síndrome de Chediak-Higashi/inmunología , Antígenos HLA-D/metabolismo , Antígenos HLA-DR/metabolismo , Lisosomas/inmunología , Vacuolas/inmunología , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular , Síndrome de Chediak-Higashi/genética , Síndrome de Chediak-Higashi/patología , Chlamydia trachomatis/inmunología , Chlamydia trachomatis/metabolismo , Coxiella/inmunología , Coxiella/metabolismo , Células HeLa , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas , Lisosomas/química , Lisosomas/microbiología , Sustancias Macromoleculares , Glicoproteínas de Membrana/análisis , Coloración y Etiquetado , Vacuolas/química , Vacuolas/microbiologíaRESUMEN
Chlamydiae replicate within an intracellular vacuole, termed an inclusion, that is non-fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, the inclusion appears to intersect an exocytic pathway from which chlamydiae intercept sphingomyelin en route from the Golgi apparatus to the plasma membrane. Chlamydial protein synthesis is required to establish this interaction. In an effort to identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several Chlamydia trachomatis inclusion membrane proteins. Microinjection of polyclonal antibodies against three C. trachomatis inclusion membrane proteins, IncA, F and G, into the cytosol of cells infected with C. trachomatis demonstrates reactivity with antigens on the cytoplasmic face of the inclusion membrane, without apparent inhibition of chlamydial multiplication. Microinjection of antibodies against the C. trachomatis IncA protein, however, results in the development of an aberrant multilobed inclusion structure remarkably similar to that of C. psittaci GPIC. These results suggest that the C. trachomatis IncA protein is involved in homotypic vesicle fusion and/or septation of the inclusion membrane that is believed to accompany bacterial cell division in C. psittaci. This proposal is corroborated by the expression of C. trachomatis and C. psittaci IncA in a yeast two-hybrid system to demonstrate C. trachomatis, but not C. psittaci, IncA interactions. Despite the inhibition of homotypic fusion of C. trachomatis inclusions, fusion of sphingomyelin-containing vesicles with the inclusion was not suppressed.