RESUMEN
Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.
Asunto(s)
Cápsulas Bacterianas/fisiología , Lipopolisacáridos/metabolismo , Manosa/metabolismo , Mycobacterium/fisiología , Animales , Cápsulas Bacterianas/metabolismo , Elementos Transponibles de ADN/genética , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Electroforesis en Gel de Poliacrilamida , Femenino , Prueba de Complementación Genética , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Interleucina-10/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Manosa/química , Manosa/fisiología , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Modelos Moleculares , Mutagénesis Insercional , Mutación , Mycobacterium/metabolismo , Infecciones por Mycobacterium/metabolismo , Infecciones por Mycobacterium/microbiología , Pez CebraRESUMEN
Protein tyrosine phosphorylation is an important mechanism of eukaryotic cell signalling which is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we report the molecular cloning of the first zebrafish protein-tyrosine phosphatase, zf-PTP-1B, the homologue of human PTP-1B. Zf-PTP-1B was catalytically active and localised to the endoplasmic reticulum, like human PTP-1B. Zf-PTP-1B was maternally expressed in zebrafish embryos, and low ubiquitous expression was detected up to day 7 of development. Microinjection of zf-PTP-1B RNA induced pleiotropic, but reproducible developmental defects. Evaluation of the live embryos at 24 h post fertilisation indicated that zf-PTP-1B induced defects in somite formation. The phenotype was dependent on protein-tyrosine phosphatase activity of zf-PTP-1B, since embryos injected with catalytically inactive zf-PTP-1B-C213S developed normally. Co-injection of wild type and inactive zf-PTP-1B led to a rescue of the zf-PTP-1B-induced phenotype, suggesting that zf-PTP-1B-C213S had dominant negative activity. The zf-PTP-1B-induced phenotype suggests that proper tyrosine phosphorylation of key proteins is essential for early development, most notably somitogenesis.