ATP6V0d2 controls Leishmania parasitophorous vacuole biogenesis via cholesterol homeostasis.

Carina Carraro Pessoa,Erica Valadares De Castro Levatti,Joyce Umbelino Da Silva Yamamoto,Fernando Real,Cristian Cortez,Luiza Campos Reis,Hiro Goto,Renato Arruda Mortara,Eduardo Milton Ramos-Sanchez,Vânia D’Almeida,Cristina Mary Orikaza,Ana Carolina Benites Badaró

doi:10.1371/journal.ppat.1007834

Abstract

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.

Highlights

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, controlling the acidification of lysosomes, endosomes, the trans-Golgi network and other intracellular vesicles [1, 2]
To approach the participation of ATP6V0d2 in cholesterol homeostasis, we have evaluated the messenger RNA (mRNA) levels of scavenger receptors and of the sterol regulatory element-binding protein 2 (SREBP2) which controls expression of genes involved in cholesterol synthesis [51], in the context of ATP6V0d2 knock-down, infection with Leishmania and treatment with oxidized low-density lipoprotein (ox-LDL)
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

Summary

Introduction

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, controlling the acidification of lysosomes, endosomes, the trans-Golgi network and other intracellular vesicles [1, 2]. The pathogen-mediated subversion of V-ATPases may involve the interference of one or several subunits that compose the two functional domains, inhibiting proton pump activity or driving V-ATPases to target different organelles. The bacterial pathogens Legionella pneumophila and Mycobacterium tuberculosis, for instance, have the ability to secrete virulence factors that directly target the H-subunit of the V1 domain of host cell V-ATPases, blocking the acidification of bacteria-containing vacuoles in which they multiply by V-ATPase exclusion [8,9,10]. Yersinia pseudotuberculosis does not exclude V-ATPases from the bacteria-containing vacuole but decreases their activity during intracellular infection [11]

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