RESUMEN
Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.
Asunto(s)
Actinas/biosíntesis , Metabolismo de los Lípidos , Macrófagos/microbiología , Mycobacteriaceae/metabolismo , Fagocitosis/fisiología , Fagosomas/microbiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Interacciones Huésped-Parásitos/efectos de los fármacos , Interacciones Huésped-Parásitos/fisiología , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Lípidos/farmacología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Fusión de Membrana/efectos de los fármacos , Fusión de Membrana/fisiología , Ratones , Mycobacteriaceae/patogenicidad , Mycobacterium avium/efectos de los fármacos , Mycobacterium avium/metabolismo , Mycobacterium avium/patogenicidad , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Óxido Nítrico/metabolismo , Fagocitosis/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P(2)-binding proteins ezrin and/or moesin were essential for this process (). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P(2), and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P(2) antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P(2) into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P(2)-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.