RESUMO
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 ΔTgCRT 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.
Assuntos
Cloroquina/farmacologia , Endossomos , Lisossomos , Proteínas de Membrana Transportadoras , Plasmodium falciparum , Proteínas de Protozoários , Toxoplasma , Toxoplasmose , Linhagem Celular , Endossomos/metabolismo , Endossomos/parasitologia , Humanos , Lisossomos/metabolismo , Lisossomos/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/genética , Toxoplasmose/metabolismo , Toxoplasmose/patologiaRESUMO
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.
Assuntos
Cloroquina/farmacologia , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Quinolinas/farmacologia , Antimaláricos/farmacologia , Transporte Biológico , Técnicas de Cultura de Células , Humanos , Malária Falciparum/genética , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Modelos Moleculares , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Isoformas de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antimaláricos/metabolismo , Cloroquina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Plasmodium vivax/metabolismo , Proteínas de Protozoários/metabolismo , Substituição de Aminoácidos , Antimaláricos/farmacologia , Transporte Biológico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cloroquina/farmacologia , Contagem de Colônia Microbiana , Resistência a Medicamentos , Humanos , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Mutagênese Sítio-Dirigida , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/crescimento & desenvolvimento , Plasmodium vivax/isolamento & purificação , Primaquina/metabolismo , Primaquina/farmacologia , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , TrítioRESUMO
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.