Abstract
Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development.
Highlights
The integral membrane protein P-glycoprotein (P-gp, MDR1, ABCB1) is one of the most studied cellular transporters of the ATP-binding cassette (ABC) transporter superfamily [1]
To analyze whether GF120918 inhibits parasite invasion, parasites were pre-treated with the inhibitor for 30 min at 37uC and allowed to infect host cells wild type (WT) or deficient in the two mouse Pgp isoforms (P-gp double knocked out (DKO)) [3] for 4 h in presence of the drug
In the present study we investigated the effects of the potent, third generation Pgp inhibitor GF120918 in the pathogenic parasite T. gondii
Summary
The integral membrane protein P-glycoprotein (P-gp, MDR1, ABCB1) is one of the most studied cellular transporters of the ATP-binding cassette (ABC) transporter superfamily [1]. The main function of P-gp is the export of xenobiotics from the cell, as corroborated by the findings that P-gp deficient mice are viable but show strikingly altered pharmacokinetics and increased sensitivity to a variety of drugs [3] In addition to this well known role, an increasing amount of evidence suggests that P-gp participates in normal physiological processes, including the transport of steroid hormones [4] and lipid translocation Indications that T. gondii P-gp may be involved in key biological processes, such as replication and host cell invasion were provided by early works using P-gp inhibitors [6,10]. GF120918 does not inhibit the P-gp-related multidrug transporters MRP1 and MRP2 [17] nor cytochrome P450 3A, a key enzyme in drug metabolism [18], and achieves adequate P-gp inhibition in vivo without significant side effects [13,19]
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