Host cell P-gp modulates parasite replication. A, WT, P-gp-deficient (DKO) and P-gp-complemented (DKO/P-gp) host cells were infected with T. gondii at multiplicity of infection 1. After 24 h p.i., intracellular parasites were quantified by direct counting. The distribution of the parasite number in single vacuoles is expressed as percentage of total vacuoles examined (n > 30). B, immunofluorescence analysis of intracellular vacuoles containing parasites at 48 h p.i. using anti-T. gondii serum, followed by fluorescein-conjugated secondary antibodies. C, quantification of parasite burden at 48 and 72 h p.i. by a colorimetric assay measuring the amount of parasite-expressed β-galactosidase. The results are expressed as percentages of parasite number in WT host cells ± S.E. (n = 6). *, p < 0.05; **, p < 0.01. D, host cell monolayers were incubated with T. gondii at a multiplicity of infection of 1. After 2 h of incubation, 20 fields of infected cells were examined, and the number of intracellular invaded parasites was determined as described under “Experimental Procedures.” The data are expressed as percentages of total parasite number ± S.E. (n = 20).

Host P-gp associates with the parasite vacuole. A, confocal microscopy of P-gp localization in P-gp-complemented (DKO/Pgp) cells infected for 24 h with T. gondii. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). PV, parasitophorous vacuole. DIC, differential interference contrast image. B, confocal microscopy of infected cells as described for A, showing P-gp (green) and parasite (red) staining. C, fluorescence microscopy of infected WT, P-gp-deficient (DKO) and P-gp-complemented (DKO/P-gp) host cells showing that T. gondii PV associated with labeled host endo-lysosomes (cholera toxin (CTX)), lysosomes (Lamp1), and Golgi (giantin). The asterisks indicate parasite vacuoles.

Host cell P-gp deficiency affects T. gondii lipid metabolism. A, quantification of free cholesterol (FC) and cholesteryl esters (CE) by enzymatic reaction (Amplex red®) in T. gondii isolated from WT or DKO host cells after four lysis passages. The data are expressed as percentages of lipid content in T. gondii from WT cells ± S.E. (n = 5). B, liquid chromatography-mass spectrometry analysis of sphingolipids of T. gondii isolated from WT or DKO host cells after four lysis passages. The data are averages ± S.E. (n = 3) of a representative from two experiments done in triplicate. Cer, ceramide; GlcCer, glucosylceramide; LacCer, lactosylceramide; CerPE, ceramide phosphoethanolamine. The predominantly detected C16 species are plotted. SM, sphingomyelin. C, left panel, TLC separation of de novo synthesized lipids labeled with [³H]palmitic acid in T. gondii isolated from WT (1) or DKO (2) cells. Right panel, newly synthesized ceramide is expressed as a percentage of ceramide in T. gondii isolated from WT host cells. The data are averages ± S.E. (n = 3).

Host cell P-gp deficiency inhibits cholesterol transport to T. gondii. A, T. gondii-infected host cells were labeled for 5 h with 0.5 μCi/ml [³H]cholesterol. The parasites were isolated, and [³H]cholesterol incorporation was measured by liquid scintillation. The data are expressed as percentages of cholesterol in parasites (dpm/10⁶ parasites) isolated from WT host cells ± S.E. (n = 6). B, host cell monolayers were infected with T. gondii for 24 h followed by incubation with NBD-cholesterol and observed by fluorescence microscopy. The asterisks indicate parasite vacuoles. C, after T. gondii invasion, DKO host cells were loaded with the indicated concentrations of cyclodextrin-cholesterol complexes, and parasite replication was monitored at 48 h p.i. as described in the legend to Fig. 1. The data are averages ± S.E. (n = 3). D, after T. gondii infection, WT and DKO host cells were treated with the class 2 amphiphile U-18666A at the indicated concentrations, and parasite replication was quantified at 48 h p.i. The results are expressed as percentages of parasite number in untreated (Cntl) WT cells. The data are averages ± S.E. (n = 6).

Cholesterol accumulates outside the parasite vacuole in absence of host P-gp. A, WT, P-gp-deficient (DKO), and P-gp-complemented (DKO/P-gp) host cell monolayers were infected with T. gondii for 24 h. After fixation, the intracellular distribution of unesterified cholesterol was visualized with filipin and observed by fluorescence microscopy. The arrows indicate parasite vacuoles. B, electron microscopy of T. gondii infected WT and P-gp-deficient (DKO) host cells at 24 h p.i., showing PV-associated mitochondria (arrowheads) and H.O.S.T. structures (arrows). Right panels, magnification of H.O.S.T. structures. Scale bars, 2 μm. C, host cells monolayers were infected with N. caninum and parasite replication quantified at 72 h p.i., as described. The results are expressed as percentages of parasite number in WT host cells ± S.E. (n = 3). D, lipids were extracted from N. caninum isolated from WT or DKO host cells after four lysis passages. A representative TLC of lipids corresponding to equal protein amount is shown. Free cholesterol (FC) and cholesteryl esters (CE) were visualized with CuSO₄. Inset, Nile red staining of cholesteryl esters-containing lipid droplets in parasites isolated from WT or DKO host cells after four lysis passages.

P-gp expression alters cholesterol metabolism in fibroblasts. A, filipin labeling of WT, P-gp-deficient (DKO), and P-gp-complemented (DKO/P-gp) host cells monolayers to visualize the intracellular distribution of unesterified cholesterol. B, host cells were labeled with [³H]cholesterol, and the time course of [³H]cholesterol esterification was measured at the indicated time points. The data are percentages of [³H]cholesteryl esters (CE) of total cellular [³H]cholesterol. C, cells were labeled with [³H]cholesterol for 24 h, washed, and incubated with 2 mm methyl-β-cyclodextrin. Medium aliquots were taken at the indicated time points, and extracted [³H]cholesterol was measured by liquid scintillation. Extracted cholesterol is expressed as a percentage of total [³H]cholesterol (medium + cell associated radioactivity). D and E, the cells were similarly labeled and incubated with 20 μg/ml reconstituted HDL (D) or 10 μg/ml apoA-I (E). Cholesterol efflux is expressed as a percentage of total [³H]cholesterol as before. F, cells were loaded with the indicated concentrations of cyclodextrin-cholesterol complexes for 24 h, and viability was measured by AlamarBlue® assay. Results are expressed as percentage of untreated (cntl) cell viability. All of the data are averages ± S.E. (n = 3).