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 organelle, 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 contribute to the maturation of 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 the TgCRT protein plays a key role in maintaining the integrity of the parasite’s endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in volume by ~15-fold and overlaps with adjacent endosome-like compartments. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in Δ TgCRT parasites. Expression of subtilisin protease 1 is significantly reduced, which impedes trimming of microneme proteins, and significantly decreases parasite invasion. Chemical or genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring integrity of the endolysosomal system, microneme protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in substrate transport that impacts VAC volume and the integrity of the endolysosomal system in T . gondii .

The Plasmodium falciparum malarial parasite genome appears to encode one and only one phosphatidylinositol 3'-kinase (PI3K), and sequence analysis suggests that the enzyme is a "class III"- or "Vps34"-type PI3K. PfVps34 has generated excitement as a possible druggable target and potentially a key target of artemisinin-based antimalarials. In this study, we optimize the PfVps34 gene for heterologous expression in yeast, purify the protein to homogeneity, use a recently validated quantitative assay for phosphatidylinositol 3'-phosphate production from phosphatidylinositol ( Hassett et al., companion paper; DOI 10.1021/acs.biochem.7b00416 ) to quantify activity and drug inhibition of that activity, and investigate the importance of key residues in the enzyme's catalytic and "N-lobe" domains. Data suggest that PfVps34 is indeed inhibited by artemisinin and related drugs but only under conditions that cleave the drugs' endoperoxide bridge to generate reactive alkylating agents.

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum malaria is widespread and has limited the use of CQ in many regions of the globe. Malaria caused by the related human parasite P. vivax is as widespread as is P. falciparum malaria and has been treated with CQ as extensively as has P. falciparum, suggesting that P. vivax parasites have been selected with CQ as profoundly as have P. falciparum parasites. Indeed, a growing number of clinical reports have presented data suggesting increased P. vivax CQR. Cytostatic (growth inhibitory) CQR for P. falciparum is caused by Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations, and it has been proposed that mutations in the PvCRT orthologue may simliarly cause P. vivax CQR via increasing CQ transport from the P. vivax digestive vacuole. Here we report the first quantitative analysis of drug transport mediated by all known mutant isoforms of Plasmodium vivax chloroquine resistance transporter (PvCRT) in order to test the protein's potential link to growing P. vivax CQR phenomena. Small, but statistically significant, differences in the transport of CQ and other quinoline antimalarial drugs were found for multiple PvCRT isoforms, relative to wild type PvCRT, suggesting that mutations in PvCRT can contribute to P. vivax CQR and other examples of quinoline antimalarial drug resistance.

Patterns of multiple amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT, UniProtKB Q8IBZ9) have previously been shown to mediate chloroquine resistance in P. falciparum malarial parasites. Recent reports suggest that novel mutations in PfCRT may mediate resistance to piperaquine (PPQ), which is used extensively as a partner drug in one prominent artemisinin combination therapy. How these novel PfCRT isoforms might mediate PPQ resistance (PPQR) is not known. Using codon optimization and other previously perfected methods for PfCRT analysis in yeast, we have expressed all known PPQR-associated PfCRT isoforms in Saccharomyces cerevisiae yeast and tested whether these isoforms catalyze PPQ transport. Relationships between relative PPQ and CQ transport are analyzed for these isoforms versus other previously recognized drug resistance-associated PfCRT isoforms.

19Toxoplasma gondii, with the ability to use foodborne, zoonotic, and congenital routes of transmission, 20 is an apicomplexan parasite that can cause severe infectious disease in the immunocompromised human 21 population. The parasites harbor a lysosome-like digestive vacuole, termed Vacuolar Compartment/Plant- 22Like Vacuole (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of 23 Toxoplasma parasites. The VAC supplies proteolytic enzymes that are required to maturate the parasite's 24 invasion effectors and digest autophagosomes and endocytosed host proteins. Our previous study 25 identified a Toxoplasma ortholog of malarial chloroquine resistance transporter that localized in the VAC. 26Here, we genetically ablate TgCRT and reveal that the TgCRT plays a key role in maintaining the integrity 27 of parasite's endolysosomal system by controlling the morphology of the VAC. The VAC dramatically 28 increases in size by ~15-fold and co-localizes with its adjacent endosome-like compartment when TgCRT 29 is absent. To reduce the aberrant swelling, the parasites decrease the transcript and protein levels of 30 endolysosomal proteases in ∆crt parasites. One subtilisin protease residing within the endolysosomal 31 system was significantly reduced at both the transcription and translation stages, which impairs the 32 trimming of micronemal proteins on the surface of the parasites, thereby decreasing parasite invasion. Our 33 study also showed that chemical and genetic inhibition of proteolysis within the VAC reduces its size and 34 partially restores the endolysosomal system, as well as invasion and micronemal protein trimming. In 35 summary, these findings reveal for the first time a native role of this CRT ortholog in controlling the size of 36 the VAC and integrity of the endolysosomal system in Toxoplasma parasites to facilitate parasite invasion 37 of host cells. These observations also indicate the function of TgCRT in mediating the transport of small 38 solutes to regulate osmotic pressure within the VAC. Our findings will help expand understanding of CRT 39 orthologs in other apicomplexan parasites. 40 41 3 Author Summary 42Toxoplasma gondii is an obligate intracellular protozoan parasite that belongs to the phylum 43 Apicomplexa and infects virtually all warm-blooded organisms. Approximately one-third of the human 44 population is infected with Toxoplasma. The parasites invade host cells by using processed invasion 45 effectors to disseminate infection. A lysosome-like digestive vacuole is involved in refining these invasion 46 effectors to reach their final forms. One malarial chloroquine resistance transporter-like protein was found 47 to span the membrane of the digestive vacuole in Toxoplasma. Although the mutated version of the 48 malarial chloroquine resistance transporter has been shown to confer resistance of chloroquine treatment 49 in malaria parasites, its native role in apicomplexan parasites remains unknown. In this study, we report 50 that this transmembrane protei...