Non-canonical activation of the ESCRT machinery is required for division of Leishmania donovani parasitophorous vacuoles and persistence

Abstract

In the mammalian host, L. donovani are intracellular pathogens that reside in vacuolar compartments (often called Leishmania parasitophorous vacuoles (LdLPVs)). LdLPVs harbor individual parasites that enigmatically divide upon replication of the parasite. In this study, we evaluated the role of the ESCRT machinery in the division of LdLPVs and parasite persistence in infected cells. We found that the ESCRT I member, TSG101 and the ESCRT III members, CHMP2B and CHMP4B are recruited to LdLPVs. In addition, Vps4a, an accessory molecule required for recycling of ESCRT III molecules is also recruited to LdLPVs. Interestingly, infection of cells expressing a dominant negative version of Vps4a that prevents the recycling of ESCRT III revealed that most LdLPVs recruit ESCRT components constitutively. Based on that finding, we proposed that the recruitment of ESCRT molecules to LdLPVs is enabled by the display of the phosphoinositide, PI(3,4)P2 on LdLPVs. To assess the functional importance of recruiting ESCRT molecules to LdLPVs, we monitored L. donovani infections in cells in which ALIX or TSG101 were knocked down. ALIX knock down resulted in LdLPVs that were distended and harbored 4 or more parasites, which is significantly different from LdLPVs in wild type macrophages that harbor at most, 2 parasites. Moreover, reduced levels of ALIX resulted in a significant reduction in parasite numbers. These findings revealed the critical role for activation of the ALIX-ESCRTIII axis in L. donovani pathogenesis. This is the first demonstration that the ESCRT machinery plays a role in the division of pseudo-organelles that harbor an intracellular pathogen. SignificanceThe endosomal sorting complex required for transport (ESCRT) machinery plays critical mechanistic roles in physiological processes including cell division (cytokinesis). It can be hijacked to promote the spread and persistence of infectious agents including in the budding of viruses and nutrient acquisition by intracellular pathogens. In this study, we uncover a new role for the ESCRT machinery in the infection of macrophages by Leishmania donovani (Ld). Within infected cells, each Ld parasite resides in a Leishmania parasitophorous vacuole (LPV) that enigmatically divides to accommodate daughter parasites. We show that a non-canonical activation of the ESCRT machinery is required for division of LPVs and for parasite persistence. Future studies on the mechanisms for selective activation of the ESCRT machinery would reveal targets for the control of this deadly pathogen.