Dual staining using polyclonal mouse anti-P-gp minigene (green) and anti-VP1 (crimson) antibodies showing incomplete co-localization of both proteins (arrows). MIC2 secretion. A. Immunoblot evaluation of supernatant (SN) and pellets of T. gondii civilizations incubated with 10 M GF120918 (GF), 1 M of Ca2+ ionophore A23187 (CaI) or solvent (cntl). Tubulin staining (Tub) was utilized as a launching control. Because of the low degrees of basal MIC2 secretion weighed against A23187- induced secretion, probed membranes had been exposed for much longer situations (asterisk) to record unsaturated indication for both protein. B. Densitometric quantification from the comparative quantity of secreted MIC2, normalized by the full total protein amount, portrayed as percentage of neglected control (cntl).(0.90 MB TIF) pone.0010062.s002.tif (880K) GUID:?20776CEC-388B-49E6-9F89-BD2D1BCD4DCA Abstract Up-regulation from the membrane-bound efflux pump P-glycoprotein (P-gp) is from the sensation of multidrug-resistance in pathogenic organisms, including protozoan parasites. Furthermore, P-gp is important in regular physiological processes, our knowledge of these P-gp features continues to be limited however. Within this research we investigated the consequences from the P-gp inhibitor GF120918 in P-gp was localized in acidocalcisomes, the main Ca2+ storage space in the parasite, on the plasma membrane, and Mouse monoclonal to mCherry Tag in the intravacuolar tubular network. Furthermore, metabolic labeling of extracellular parasites uncovered that inhibition or down-regulation of P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of P-gp in essential processes of the parasite Isochlorogenic acid A biology and further validate the potential of P-gp activity as a target for drug development. Introduction 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 clinical importance of P-gp derives from the fact that over-expression of this transporter is commonly associated with the phenomenon of multidrug resistance [2], a major public health problem derived from drug-resistant cancer cells and microbial pathogens. 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 now suggests that P-gp also participates in normal physiological processes, including the transport of steroid hormones [4] and lipid translocation (rev. in [5]). Here we investigated the effects of the potent P-gp inhibitor GF120918 in the biology of 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]. However, given that these studies used host cells containing P-gp, it was not possible to discriminate between the contribution of and host cell P-gp. Indeed, we recently showed that host cell P-gp plays a crucial role in replication by facilitating the transport of host cholesterol to the parasite vacuole [11]. In this study we used P-gp deficient Isochlorogenic acid A host cells [3] in parallel with pharmacological inhibition of Isochlorogenic acid A P-gp, thereby enabling more selective insights into the specific role of P-gp. Inhibition of parasite P-gp was achieved with the acridonecarboxamide derivative GF120918, a potent competitive P-gp inhibitor of the latest generation [12], [13], whose use has been widely published both without significant side effects [13], [19]. Results GF120918 inhibits parasite invasion As an obligate intracellular parasite, depends completely on host cells for its survival and propagation; thus host cell invasion is an essential process in the parasite’s biology. To analyze whether P-gp inhibition compromises parasite invasion, we blocked P-gp function in isolated parasites with GF120918, a potent P-gp inhibitor of the latest generation [13]. GF120918 was found to strongly hamper P-gp function in the parasite at low micromolar concentrations, as assessed by efflux analysis of the specific P-gp substrate rhodamine 123 (Fig. 1A). To analyze whether GF120918 inhibits parasite invasion, parasites were pre-treated with the inhibitor for 30 min at 37C and allowed to infect host cells wild type (WT) or deficient in the two mouse P-gp isoforms (P-gp DKO) [3] for 4 h in presence of the drug. GF120918 was then removed and the infection was determined by counting the parasite vacuoles after 24 h incubation. GF120918 treatment reduced the number of intracellular vacuoles by 50% in both host cell types, indicating that host P-gp is not involved in parasite invasion (Fig. 1B, white bars). Importantly, the invasion inhibition was not caused by parasite lethality following compound treatment, as GF120918 did not significantly compromise parasite viability at the concentration inhibitory for invasion (Fig. 1F). To analyse whether the presence of GF120918 at the time of infection was necessary for the inhibitory effect, parasites were pre-treated with GF120918, washed and incubated with host cells in absence of the drug. Also in these experimental.