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Mutational Analysis of a Putative Drug Resistance Transporter in the Human Parasite T. gondii
Author(s) -
RiveraLugo Rafael,
Dou Zhicheng,
Carruthers Ver
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.574.16
Subject(s) - biology , toxoplasma gondii , cysteine , apicoplast , intracellular parasite , proteases , serine , virology , plasmodium falciparum , gene , genetics , biochemistry , malaria , immunology , apicomplexa , enzyme , antibody
Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in animals and humans. At least 30% of the human population is infected. The infection can lead to lethal disease in immunocompromised individuals including AIDS and organ‐transplant patients. No existing drugs are capable of eradicating the parasites from infected people. Opening new options, we recently reported that Toxoplasma parasites intercept host cytosolic proteins by funneling them into a lysosome‐equivalent organelle termed vacuolar compartment (VAC) where they are digested. Additional work revealed that the VAC harbors a homolog of the malaria chloroquine resistance transporter (CRT). A single point mutation in CRT confers resistance to chloroquine treatment for malaria, profoundly impacting fatalities. Knocking out the CRT gene in Toxoplasma results in an enlarged VAC and reduced fitness. Topology analysis of the CRT primary sequence showed that four cysteine residues localize to a loop oriented in the lumen of the VAC. We hypothesized that these conserved cysteine residues function to regulate redox potential of the VAC and/or bind to metal ions to optimize the activity of VAC proteases. We performed site‐directed mutagenesis to mutate these four cysteine amino acids to serine residues individually and introduce them into CRT‐deficient parasites. We found that Δ crtCRT C585S and Δ crtCRT C596S did not restore the size of the VAC, and that Δ crtCRT C609S did restored it but about 50% of the parasites showed mistrafficking of CRT. By identifying important conserved residues in a therapeutically relevant transporter this work forms a basis for future mechanistic dissection of CRT function