RESPONSE OF CULTURED MACROPHAGES TO MYCOBACTERIUM TUBERCULOSIS, WITH OBSERVATIONS ON FUSION OF LYSOSOMES WITH PHAGOSOMES

Abstract

The cytological response to the ingestion of tubercle bacilli by cultured mouse peritoneal macrophages has been studied by electron microscopy. Methods included a quantitative assessment based on systematic surveying of cell profiles, and of phagosomes and their contained bacteria, encountered in thin sections; classification of the sectioned bacteria into visibly damaged and apparently intact categories; prelabeling of dense granules (secondary lysosomes) with ferritin as an aid to identifying the occurrence and frequency of phagosome-lysosome fusion; and monitoring of bacterial growth and viability by light microscopy and cultural counts. The situations studied were as follows: progressive infection with the multiplying virulent strain H37Rv; ingestion of the same strain previously inactivated by gamma radiation; infection with an attenuated strain (BCG); and a stabilized virulent infection induced by the surfactant Macrocyclon. In the bacterial suspensions used routinely for inoculation, about half the bacilli were viable, matching closely the proportions of intact and damaged organisms identified with the electron microscope. In the inoculated macrophages, some phagosomes containing intact bacilli and others containing damaged bacilli were always to be found; but the proportion of organisms scored as damaged increased, and that of intact organisms decreased, in situations where the population as a whole had been rendered nonviable before inoculation, or where they became so intracellularly as in the late stages of a BCG infection. Evidence of fusion of ferritin-marked lysosomes with some bacterium-containing phagosomes was obtained in all experiments, but a significant difference was regularly observed according to whether the bacilli were damaged or intact. Virtually all phagosomes containing damaged bacilli showed signs of fusion; but when many phagosomes were present containing apparently intact organisms (as with actively multiplying strain H37Rv or with this strain held at a steady level of viability by Macrocyclon, and also with strain BCG at an early stage of that infection), signs of fusion of lysosomes with these phagosomes were infrequent. From these findings it is inferred that intracellular survival of M. tuberculosis in cultured macrophages is associated with a tendency to nonfusion of dense granules with the phagosome, thus avoiding direct exposure of the bacilli to the contents of these organelles. It is suggested, further, that fusion of dense granules with the phagosome, leading to digestion, is determined by recognition of the bacillus as nonviable. The possibility is discussed that the cytological response to different mycobacterial infections may reflect differences of a basic nature between facultative and obligate intracellular parasitism.

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

“…The ability to arrest phagosome maturation is a hallmark of M. tuberculosis (Armstrong & Hart, 1971). Besides avoidance of phago-lysosomal fusion, relatively little is known about how M. tuberculosis manages its intracellular lifestyle.…”

“…It involves acquisition, loss and modification of defined host proteins, and ultimately results in acidification of the phagosomal lumen to pH ,5 and generates an environment in which microbes are exposed to lytic enzymes and reactive oxygen and nitrogen intermediates (Huynh & Grinstein, 2007;Schnappinger et al, 2003;Vieira et al, 2002). Pathogenic mycobacteria interfere with the phagosome maturation process (Armstrong & Hart, 1971;Clemens & Horwitz, 1995;Hasan et al, 1997;Via et al, 1997;Xu et al, 1994). The mycobacterial phagosome retains the early endosomal GTPase rab5 (Clemens et al, 2000;Kelley & Schorey, 2003;Via et al, 1997) and access to transferrin within the rapid recycling pathway (Clemens & Horwitz, 1996;Sturgill-Koszycki et al, 1996).…”

Surface properties of bacillus Calmette-Guérin-activated mouse macrophages. Reduced expression of mannose-specific endocytosis, Fc receptors, and antigen F4/80 accompanies induction of Ia.

“…Quantitative assessments of the synthesis, internalization, turnover, and possible recycling of all the molecules concerned should yield further insights into the cellular mechanisms responsible. It should also be noted that uptake of BCG organisms is known to inhibit phagolysosomal fusion (31) and to reduce phagocytosis of various particulates, with or without opsonins (23). In our studies, the peritoneal macrophages • obtained after infection contained few detectable organisms and the surface changes in these cells could not be mimicked in uninfected cells by uptake of BCG organisms in vitro.…”

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

“…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).…”