The ortholog of chloroquine resistance transporter (TgCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion

Abstract

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like digestive vacuole, termed the \"Vacuolar Compartment/Plant-Like Vacuole\" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that are required to mature the parasites invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that TgCRT protein plays a key role in maintaining the integrity of the parasites endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in size by ~15-fold and co-localizes with its adjacent endosome-like compartment. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in {Delta}TgCRT parasites. Expression of one endolysosomal subtilisin protease is quite significantly reduced, which impedes trimming of micronemal proteins, and significantly decreases parasite invasion. Chemical and genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring the endolysosomal system, micronemal protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in controlling VAC volume and the integrity of the endolysosomal system in T. gondii.\n\nAuthor SummaryToxoplasma gondii is an obligate intracellular protozoan parasite that belongs to the phylum Apicomplexa and that infects virtually all warm-blooded organisms. Approximately one-third of the human population is infected with Toxoplasma. The parasites invade host cells via processed invasion effectors in order to disseminate infection. A lysosome-like digestive vacuole (VAC) is involved in refining these invasion effectors to reach their final forms. A T. gondii ortholog of the malarial chloroquine resistance transporter protein (TgCRT) was found to be localized to the VAC membrane. Although the mutated version of the malarial chloroquine resistance transporter (PfCRT) has been shown to confer resistance to chloroquine treatment, its physiologic function remains poorly understood. Comparison between the related PfCRT and TgCRT proteins facilitates definition of the physiologic role of CRT proteins. In this study, we report that TgCRT plays a key role in regulating the integrity and proteolytic activity of the VAC and adjacent organelles, the secretion of invasion effectors, and parasite invasion and virulence. To relieve osmotic stress caused by VAC swelling when TgCRT is deleted, parasites repress proteolytic activities within this organelle to decrease solute accumulation, which then has secondary effects on parasite invasion. Our findings highlight a common function for PfCRT and TgCRT proteins in regulating apicomplexan parasite vacuolar size and function.