Date Published: June 14, 2019
Publisher: Public Library of Science
Author(s): Carina Carraro Pessoa, Luiza Campos Reis, Eduardo Milton Ramos-Sanchez, Cristina Mary Orikaza, Cristian Cortez, Erica Valadares de Castro Levatti, Ana Carolina Benites Badaró, Joyce Umbelino da Silva Yamamoto, Vânia D’Almeida, Hiro Goto, Renato Arruda Mortara, Fernando Real, Albert Descoteaux.
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.
Vacuolar H+-ATPases (V-ATPases) are membrane-associated ATP-dependent multimeric enzymes responsible for pumping protons from the cytosol into the lumen of intracellular organelles, thus controlling the acidification of lysosomes, endosomes, the trans-Golgi network and other intracellular vesicles [1, 2]. V-ATPases display two functionally distinct domains composed of several subunits: the cytosolic domain V1, composed of eight subunits (A, B, C, D, E, F, G and H) and that is implicated in ATP hydrolysis, and membranal domain V0, which is composed of subunits a, d, e, c, c’, and c” and is implicated in proton transport across the vesicle membrane .
We report the participation of an alternative isoform of the V-ATPase subunit d, the isoform d2 (ATP6V0d2) in controlling the biogenesis of pathogen-containing vacuoles generated by L. amazonensis in macrophages. ATP6V0d2, whose expression is restricted to certain cell lineages, including macrophages, does not participate in phagolysosome acidification, indicating that the ubiquitous isoform d1 (ATP6V0d1) participates exclusively in the canonical function of this V-ATPase, while isoform d2 switches the V-ATPase toward noncanonical, acidification-independent functions, such as membrane fusion, regulation of lysosome enzymatic activities and downregulation of macrophage inflammatory burst [4, 21, 24, 56]. Therefore, the variant ATP6V0d1 is still expressed in ATP6V0d2 knock-down macrophages (ATP6V0d2-KD), capable of composing functional V-ATPases that acidify phagolysosomes and parasite-containing vacuoles. The preservation of phagolysosome acidification in the absence of the d2 variant demonstrated by us here and by others [21, 24] is a solid evidence that V-ATPases in ATP6V0d2-KD macrophages are functional and thus composed of all subunits required for their canonical functions.