ATP6V0d2 controls Leishmania parasitophorous vacuole biogenesis via cholesterol homeostasis.

V-ATPases are part of the membrane components of pathogen-containing vacuoles, although their function in intracellular infection remains elusive. In addition to organelle acidification, V-ATPases are alternatively implicated in membrane fusion and anti-inflammatory functions controlled by ATP6V0d2, the d subunit variant of the V-ATPase complex. Therefore, we evaluated the role of ATP6V0d2 in the biogenesis of pathogen-containing vacuoles using ATP6V0d2 knock-down macrophages infected with the protozoan parasite Leishmania amazonensis. These parasites survive within IFNγ/LPS-activated inflammatory macrophages, multiplying in large/fusogenic parasitophorous vacuoles (PVs) and inducing ATP6V0d2 upregulation. ATP6V0d2 knock-down decreased macrophage cholesterol levels and inhibited PV enlargement without interfering with parasite multiplication. However, parasites required ATP6V0d2 to resist the influx of oxidized low-density lipoprotein (ox-LDL)-derived cholesterol, which restored PV enlargement in ATP6V0d2 knock-down macrophages by replenishing macrophage cholesterol pools. Thus, we reveal parasite-mediated subversion of host V-ATPase function toward cholesterol retention, which is required for establishing an inflammation-resistant intracellular parasite niche.

Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis.

The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol.