Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

Abstract

Background Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. Methodology/Principal Finding In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. Conclusion/Significance Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.

Mechanism of polarized lysosome exocytosis in epithelial cells

“…S6), in agreement with data from endothelial cells (Predescu et al, 2005). Recent reports show that in fibroblasts and cardiomyocytes, cholesterol depletion by cyclodextrins increases ionomycin-induced lysosome secretion (Chen et al, 2010;Hissa et al, 2012). Conversely, cholesterol accumulation in late endosomes and lysosomes paralyzes late endocytic traffic by a variety of mechanisms including inhibition of rabGTPases, which can interfere with the recruitment of molecular motors to lysosomes, and thus reduce the population of pre-docked lysosomes at the plasma membrane, and abnormal sequestration of SNARE proteins, which would interfere with the fusion process (Fraldi et al, 2010;Lebrand et al, 2002).…”

Understanding the role of cholesterol in cellular biomechanics and regulation of vesicular trafficking: The power of imaging

“…In the light of these works, it seemed plausible that lysosomes exocytosis, induced upon cholesterol sequestration from plasma membrane, could be also triggered by cell cytoskeleton reorganization. In our work we showed that cholesterol sequestration from plasma membrane led to the exocytosis of a specific pool of lysosomes localized at the cell cortex, as seen by lysosomal cell distribution before and after treatment (lysosomal dispersion assay) [50]. This could represent docked lysosomes in which actin dynamics, induced by cholesterol sequestration, could be working to push these organelles into closer contact with plasma membrane activating the exocytic machinery (Fig.…”

“…We first thought that cholesterol sequestration, and consequently rafts disruption, was compromising lysosome fusion with host cell plasma membrane, such as previously reported for other secretory vesicles, and thus decreasing parasite invasion. However we identified that membrane cholesterol disruption was, on the contrary, inducing lysosome fusion events [50]. The lysosomal exocytic events triggered by cholesterol sequestration were independent of calcium or the lysosomal calcium sensor, Syt-VII, indicating they were unregulated events, such as the ones observed by Zamir and collegaues in cholesterol depleted axons from crayfish neuromuscular junctions [114].…”

“…The lysosomal exocytic events triggered by cholesterol sequestration were independent of calcium or the lysosomal calcium sensor, Syt-VII, indicating they were unregulated events, such as the ones observed by Zamir and collegaues in cholesterol depleted axons from crayfish neuromuscular junctions [114]. Another interesting aspect was that it occurred specifically from a pool of lysosomes localized closer to the cell plasma membrane, which we later identified as a pool recruited during parasite-host cell invasion [50,52].…”

“…They not only observed extreme re-organization of the actin cytoskeleton with an increase in stress fiber formation, but they were able to show that these effects of cholesterol were due to the activation of two pathways linked to src activation: RhoA and ROCK activation, as well as Cav-1 phosphorlation. Later, our group also showed that cholesterol sequestration induced stress fiber formation in neonatal murine primary cardiomyocytes (CM) [50]. Figure 3B shows some representative images of fibroblasts treated or not with MβCD and the effects of the treatment on actin cytoskeleton organization.…”