RESUMEN
Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.
Asunto(s)
Interacciones Huésped-Patógeno , Macrófagos/microbiología , Ácidos Micólicos/metabolismo , Fagosomas/microbiología , Rhodococcus equi/patogenicidad , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Infecciones por Actinomycetales/inmunología , Infecciones por Actinomycetales/microbiología , Animales , Línea Celular , Elementos Transponibles de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Rhodococcus equi/genética , Rhodococcus equi/inmunología , Análisis de Secuencia de ADN , VirulenciaRESUMEN
Phagocytosis is a receptor-mediated process for sequestration and inactivation of infectious microbes. It can be triggered by microbial surface compounds or particle-attached host proteins. We monitored the effector functions of murine bone marrow-derived macrophages (BMMs) in response to polystyrene-streptavidin beads coated with the defined ligands IgG1, ß-glucan, mannan, complement factors C1q or iC3b, or fibronectin (FN). Cell-autonomous effector mechanisms (uptake, phagosome maturation, cytokine responses and killing activity) were differentially triggered. All particle-ligand complexes stimulated the release of nitric oxide, but only beads coated with IgG, complement factors or FN caused production of superoxide. Beads coated with C1q, iC3b or FN strongly stimulated the secretion of pro-inflammatory TNF-α, IL-6, and IL-1ß and also of anti-inflammatory IL-10. Escherichia coli coated with C1q, iC3b or FN was killed much less efficiently than with any of the other ligands, depending on the presence of IL-10 activity. This indicated an important role of IL-10 as regulator of cell-autonomous immune functions of macrophages. Our data show that defined ligands on microbial surfaces are interesting candidates to activate innate defense mechanisms selectively and specifically.