Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii

Abstract

Lipid flipping in the membrane bilayers is a widespread eukaryotic phenomenon that is catalyzed by assorted P4-ATPases. Its occurrence, mechanism, and importance in apicomplexan parasites have remained elusive, however. Here we show that Toxoplasma gondii, an obligate intracellular parasite with high clinical relevance, can salvage phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) but not phosphatidylcholine (PtdCho) probes from its milieu. Consistently, the drug analogs of PtdCho are broadly ineffective in the parasite culture. NBD-PtdSer imported to the parasite interior is decarboxylated to NBD-PtdEtn, while the latter is not methylated to yield PtdCho, which confirms the expression of PtdSer decarboxylase but a lack of PtdEtn methyltransferase activity and suggests a role of exogenous lipids in membrane biogenesis of T. gondii. Flow cytometric quantitation of NBD-probes endorsed the selectivity of phospholipid transport and revealed a dependence of the process on energy and protein. Accordingly, our further work identified five P4-ATPases (TgP4-ATPase1-5), all of which harbor the signature residues and motifs required for phospholipid flipping. Of the four proteins expressed during the lytic cycle, TgP4-ATPase1 is present in the apical plasmalemma; TgP4-ATPase3 resides in the Golgi network along with its noncatalytic partner Ligand Effector Module 3 (TgLem3), whereas TgP4-ATPase2 and TgP4-ATPase5 localize in the plasmalemma as well as endo/cytomembranes. Last but not least, auxin-induced degradation of TgP4-ATPase1-3 impaired the parasite growth in human host cells, disclosing their crucial roles during acute infection. In conclusion, we show selective translocation of PtdEtn and PtdSer at the parasite surface and provide the underlying mechanistic and physiological insights in a model eukaryotic pathogen.

Keywords: Lem3/Cdc50; NBD-lipid; P4-ATPase; phosphatidylethanolamine; phosphatidylserine.

Figure 1

Toxoplasma gondii can salvage PtdSer and PtdEtn from the extracellular environment. Tachyzoites were incubated with NBD-lipids (37 °C, 30 min), followed by microscopic imaging, flow cytometry, and thin-layer chromatography. A, imaging of parasites immediately after incubation with NBD-conjugated PtdEtn, PtdSer, PtdCho, or sphingomyelin. Cells were examined by fluorescence microscopy and differential interference contrast (DIC). The scale bar represents 10 μm. B, geometric mean fluorescence intensity of NBD-labeled tachyzoites, as determined by flow cytometry (n = 3; mean ± SE). C, TLC-resolved lipid profiles of parasites treated with indicated probes. Lipid samples were resolved onto a silica plate along with standards (Std.) and imaged for fluorescence. NBD-FFA, NBD-linked free fatty acids. NBD, nitrobenzoxadiazole; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine.

Figure 2

Lipid internalization in T. gondii is a temperature-, ATP-, and protein-driven process. Tachyzoites were incubated with NBD-lipids (30 min) followed by flow cytometric analysis. A, effect of temperature on the import of NBD-lipids by extracellular tachyzoites. B, ATP-dependence of lipid uptake. For the ATP content in corresponding samples (run alongside) refer to Figure S2C. C, influence of N-ethylmaleimide (NEM) treatment on the import of NBD-lipids. Data shown here are normalized to NBD-PtdEtn samples (set as 100%) at 37 °C (A), ATP-replete (B), or NEM-treatment (C) conditions. A–C, illustrate means ± SE from three assays (∗∗∗p ≤ 0.001).

Figure 3

T. gondii encodes five potential phospholipid flippases.A, primary structure of TgP4-ATPase1-5 encoded by the parasite. Functional domains were predicted by NCBI conserved domain search, PFAM v32.0 and SMART programs, and the number and location of transmembrane helices are based on the TMHMM, TMpred, and Phobius suites. B, a single parsimonious cladogram depicting the relationship of TgP4-ATPase1-5 with homologs from H. sapiens, S. cerevisiae, and C. elegans. Colored bars indicate a classification of P4-ATPases, and dots on branch nodes show the bootstrap values. The bar scale signifies the amino acid substitutions per site. NCBI Accession: TgP4-ATPase1, MT268297; TgP4-ATPase2, MT268298; TgP4-ATPase3, MT268299; TgP4-ATPase4, MT268300; TgP4-ATPase5, MT268301; HsATP8A1, Q9Y2Q0; HsATP8A2, Q9NTI2; HsATP8B1, O43520; HsATP8B2, P98198; HsATP8B3, O60423; HsATP8B4, Q8TF62; HsATP9A, O75110; HsATP9B, O43861; HsATP10A, O60312; HsATP10B, O94823; HsATP10D, Q9P241; HsATP11A, P98196; HsATP11B, Q9Y2G3; HsATP11C, Q8NB49; ScDrs2, P39524; ScNeo1, P40527; ScDnf1, P32660; ScDnf2, Q12675; ScDnf3, Q12674; CeTAT-1, Q9U280; CeTAT-5, G5EBH1.

Figure 4

P4-ATPases expressed in tachyzoite exhibit different subcellular distribution.A, 3′-Insertional tagging (3′IT) of TgP4-ATPase1-5 genes with 10xHA epitope a.k.a. spaghetti monster HA (smHA). Individual constructs encoding for Cas9 and gene-specific sgRNA were cotransfected with PCR amplicons comprising smHA and HXGPRT selection cassette (S.C.) into the RHΔku80-hxgprt⁻ strain. Transgenic parasites were drug-selected and cloned by limiting dilution for further assays. B, genomic PCR screening to show the occurrence of homologous recombination in clonal transgenic strains expressing smHA-tagged TgP4-ATPase1-5. Location of the screening primer pair is denoted in (A). Genomic DNA of the parental RHΔku80-hxgprt⁻ strain was included as a negative control. The letters “T” and “P” denote transgenic and parental strains, respectively. C, immunofluorescence of intracellular parasites expressing either of the five TgP4-ATPase-smHA_3′IT. Tachyzoites replicating in human foreskin fibroblast cells were fixed 24 h post infection and immunostained with α-HA and α-TgGap45 antibodies. D, costaining of TgP4-ATPase1 and TgP4-ATPase3 with selected organelle markers. Parasites expressing TgP4-ATPase1-smHA_3′IT were stained with α-HA and α-TgIsp1 antibodies. To colocalize TgP4-ATPase3-HA_3′IT, parasites were transfected with a plasmid expressing TgErd2-Ty1 and costained using α-HA and α-Ty1 antibodies. In (C and D), DAPI represented by blue in merged pictures was used for nuclei staining. E, immunoblot of parasites encoding TgP4-ATPase-smHA_3′IT proteins. The protein extract prepared from extracellular tachyzoites (10⁷) were resolved by 6% SDS-PAGE, followed by immunoblotting using α-HA and α-TgHsp90 (loading control) antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

P4-ATPase1-2 and 5 localize in the plasma membrane. A, subcellular distribution of chosen TgP4-ATPase-smHA_3IT proteins after the drug-induced splitting of the plasmalemma and inner membrane complex (IMC). Parasites were incubated with α-toxin (20 nM, 3 h) before staining for the HA tag in combination with the antibodies recognizing IMC (TgGap45) or plasma membrane (TgSag2). B, experimental testing of the C-terminal orientation in TgP4-ATPase1-smHA_3IT protein. Tachyzoites were immunostained after treating with detergent-free PBS (“Intact”) or detergent-PBS (“Permeabilized”). The parasite and host-cell nuclei were visualized by DAPI (blue) in A-B. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6

Knockdown of TgP4-ATPase1-3 is detrimental to the tachyzoite growth. A, scheme for CRISPR-Cas9–assisted 3′-insertional tagging of TgP4-ATPase1-3 with an auxin-inducible degron (AID) and 3xHA. pU6-Cas9-TgP4-ATPaseX_sgRNA plasmids (encoding Cas9 and gene-specific sgRNA) were cotransfected with matching donor amplicons (AID-3xHA-3′UTR_Gra1-DHFR-TS) flanked by a 40-bp crossover sequence (COS) into a parental strain (RHΔku80-hxgprt⁻-TIR1), and drug-selected for the DHFR-TS selection cassette (S.C.) using pyrimethamine. Transgenic tachyzoites (P_native-TgP4-ATPaseX-AID-3xHA_3′IT-3′UTR_Gra1, X = 1–3) expressed AID-3xHA-tagged TgP4-ATPases, which could be conditionally regulated by auxin. B, verification of the Cas9-assisted AID-3xHA tagging of genes in mutants by PCR screening using specific primer pairs (see A). The letters “T” and “P” denote transgenic and parental strains, respectively. C, auxin-dependent expression of AID-3xHA-tagged TgP4-ATPase proteins. Parasites were cultured with or without 500 μM IAA for 24 h and then stained with α-HA (green) and α-TgGap45 (red) antibodies and DAPI (blue). D, immunoblots of transgenic strains before and after auxin treatment. Equivalent amounts of protein for each sample were resolved on 6% SDS-PAGE, followed by blotting and immunostaining of HA and TgHsp90 (loading control). The asterisks denote the size of 250, 200, and 170 kDa for epitope-tagged forms of TgP4-ATPase1-3, respectively. Note the difference in the size of TgP4-ATPase1-AID-3xHA in comparison with the smHA-tagged form, which did not reveal a band of 250 kDa (Fig. 4E). E, plaques formed by the mutant and parental strains in the absence or presence of IAA. Crystal violet–stained images reveal plaques formed by successive lytic cycles of the stated strains. The plaque area, scored by ImageJ, is depicted in arbitrary units (a. u.). About 150 to 200 plaques of each strain were evaluated (n = 3 assays; means ± SE; ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001). DAPI, 4′,6-diamidino-2-phenylindole; DHFR-TS, dihydrofolate reductase–thymidylate synthase.

Figure 7

TgP4-ATPases expressed in tachyzoites of T. gondii.A, predicted topologies of TgP4-ATPase proteins. The M (membrane), A (actuator), P (phosphorylation), and N (nucleotide-binding) domains are present in all five parasite flippases, but the domain R (regulatory) occurs only in TgP4-ATPase1-2. B, Subcellular distribution of TgP4-ATPase1-3 and 5 proteins in the tachyzoite stage. TgP4-ATPase1 localizes in the apical plasma membrane, whereas TgP4-ATPase3 and its noncatalytic β subunit (TgLem3) reside in the Golgi network. TgP4-ATPase2 and TgP4-ATPase5 are distributed in the plasmalemma and endo/cytomembranes. TgP4-ATPase4 is not expressed in the tachyzoite stage. Import of PtdSer and PtdEtn is mediated by surface-resident flippases in an ATP-dependent manner. According to the shown model, PtdSer salvaged from the external milieu can be decarboxylated by TgPSD1_mt to produce PtdEtn, likely in the mitochondrion. Note that decarboxylation reaction can also happen, at least in part, in the parasite milieu by secreted TgPSD1_pv. PSD, phosphatidylserine decarboxylase.