Induction of Mycobacterium avium growth restriction and inhibition of phagosome–endosome interactions during macrophage activation and apoptosis induction by picolinic acid plus IFNγ

Abstract

Treatment of mouse macrophages with picolinic acid (PA) and c-interferon (IFNc) led to the restriction of Mycobacterium avium proliferation concomitant with the sequential acquisition of metabolic changes typical of apoptosis, mitochondrial depolarization, annexin V staining and caspase activation, over a period of up to 5 days. However, triggering of cell death by ATP, staurosporine or H 2 O 2 failed to affect mycobacterial viability. In contrast to untreated macrophages where extensive interactions between phagosomes and endosomes were observed, phagosomes from treated macrophages lost the ability to acquire endosomal dextran. N-Acetylcysteine was able to revert both the anti-mycobacterial activity of treated macrophages as well as the block in phagosome-endosome interactions. The treatment, however, induced only a minor increase in the acquisition of lysosomal markers, namely Lamp-1, and did not increase to any great extent the acidification of the phagosomes. These data thus suggest that the anti-mycobacterial activity of PA and IFNc depends on the interruption of intracellular vesicular trafficking, namely the blocking of acquisition of endosomal material by the microbe. INTRODUCTIONMycobacterium avium is an opportunistic pathogen that infects patients suffering from local or systemic deficiencies of the immune system (Falkinham, 1996). Resistance to infection is dependent on both innate mechanisms and acquired immunity through the development of antigenspecific CD4 + T cells (Appelberg, 1994;Holland, 1996). M. avium replicates inside the macrophage and is particularly resistant to the oxidative antimicrobial mechanisms of this phagocyte (Gomes & Appelberg, 2002;Gomes et al., 1999a). Like other pathogenic mycobacteria, M. avium has evolved survival strategies that interfere with the normal intracellular trafficking of vesicular compartments. After being internalized by macrophages, mycobacteria inhibit the fusion of the vacuole they inhabit with lysosomes, thus blocking maturation of the phagosomes into phagolysosomes and proliferating in vacuoles with early endosomal characteristics (Armstrong & d'Arcy-Hart, 1971;Clemens & Horwitz, 1995;Frehel et al., 1986;Russell et al., 1997). Several mechanisms associated with this inhibition have been described, such as reduced phagosomal acidification (Crowle et al., 1991;Sturgill-Koszycki et al., 1994;de Chastellier et al., 1995) due to exclusion of the vacuolar H + -ATPase , altered signalling pathways (Malik et al., 2001;Fratti et al., 2003), interference with the recycling of Rab proteins (Via et al., 1997), retention of the actin-binding coronin/TACO protein (Ferrari et al., 1999; see also Schuller et al., 2001, for a critical reappraisal of this issue), disorganization of the actin filament network (Guérin & de Chastellier, 2000) and tight apposition of the membrane vacuole to the surface of the pathogen, leading to altered exchange of fusion factors (de Chastellier & Thilo, 1997). However, mycobacterial phagosomes keep their ability to interact extensively wi...

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