P-glycoprotein (P-gp) is usually a membrane-bound efflux pump that actively exports

P-glycoprotein (P-gp) is usually a membrane-bound efflux pump that actively exports a wide range of compounds from your cell and is associated with the phenomenon of multidrug resistance. fibroblasts. Collectively our SU6668 findings reveal a key and previously undocumented role of P-gp in host-parasite conversation and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. Introduction P-glycoprotein (P-gp ABCB1 MDR1)2 is one of the most intensively analyzed users of the ABC transporter superfamily. With remarkably broad substrate acknowledgement P-gp drives the ATP-dependent efflux of SU6668 harmful metabolites and xenobiotics from your cell (1) and is thus a central mediator of drug bioavailability. Importantly P-gp overexpression following drug treatment is responsible for the multidrug resistance (MDR) phenotype a major reason for chemotherapy failure not only in malignancy cells (2) but also in pathogenic microorganisms (3 4 Aside from its well known role in drug efflux P-gp is also expressed at basal levels in many different tissues yet the normal physiological functions of the protein remain poorly comprehended. The possibility that physiological levels of host P-gp play a role in host-pathogen conversation other than mediating drug resistance has not been investigated so far. We resolved this question using as a model pathogenic parasite. is the causative agent of toxoplasmosis a potentially fatal disease not only for immunocompromised patients and fetuses but according to SU6668 recent insights also emerging as a life threatening contamination in immunocompetent individuals (5). infects virtually all nucleated host cells and resides in a highly specialized vacuole called the parasitophorous vacuole (PV) which is usually created by invaginating the host cell membrane at the time of invasion. The PV is not qualified for lysosome fusion thus avoiding acidification (6) but it is usually closely associated with host organelles including lysosomes mitochondria and endoplasmic reticulum (examined in Ref. 7). Even though the PV does not intersect directly with host vesicular traffic remains dependent on host SU6668 cells for a number of critical nutrients. Significant progress has been made in our understanding of the mechanisms uses to scavenge nutrients from its host especially in the case of lipid molecules. An important recent example was the identification of H.O.S.T. (host organelle-sequestering tubulo-structures) a unique system of tubular structures formed by the parasite to sequester cholesterol-containing endo-lysosomes from your host cytoplasm into the PV (8). However the molecular mechanisms of the traffic from your host cell to the PV are not completely elucidated and the presence of transporters has been proposed frequently. To analyze whether the P-gp transporter plays a role in biology ZNF914 we compared parasite replication in wild type (WT) mouse embryonic fibroblasts with double knock-out (DKO) fibroblasts in which neither of the two murine P-gp isoforms are expressed (9). In parallel we also analyzed DKO cells complemented with the human P-gp homologue (DKO/P-gp) (10) which restored P-gp functionality to DKO cells and allowed P-gp expression levels higher than those found in WT cells (supplemental Fig. S1). In this way our model did not depend on either drug-selected P-gp-overexpressing cells which may acquire adaptation mechanisms different from P-gp overexpression during the development of the MDR phenotype or P-gp inhibitors several of which are known to have side effects on host metabolism. EXPERIMENTAL PROCEDURES Biochemical Reagents Unless normally stated all chemicals were purchased from Sigma cell culture reagents were from Invitrogen and radiolabeled lipids were from Amersham Biosciences. Anti-P-gp monoclonal antibody C219 was purchased from Alexis Biochemicals; anti-Lamp1 1D4B antibody was obtained through the Developmental Studies Hybridoma Lender (University or college of Iowa Iowa City IA); anti-giantin and anti-tubulin were a kind gift from J. Rohrer and M. A. Hakimi respectively. Conjugated secondary antibodies were from Invitrogen. Reconstituted high density lipoproteins and apolipoprotein A-I (apoA-I) were a kind gift from P. Lerch (CSL Behring Bern Switzerland). NDB-cholesterol was from Avanti Polar Lipids. Mammalian Cell and Parasite Culture Mouse embryonic fibroblasts double knocked out for P-gp (77.1 Mdr1a?/?/Mdr1b?/?) (9) triple knocked out for P-gp and MRP1 (3.8 Mdr1a?/?/Mdr1b?/?/Mrp1?/?) (11) and parental.