Quantitative analysis of polar lipids with masses less than 1000, including diacyl, ether-linked (ePC; i.e. alk(en)yl/acyl PC), and lyso PC, diacyl, ether-linked (ePE), and lyso PE, diacyl and ether-linked (ePS) PS, diacyl PI, diacyl PA, SM, PE-cer, MHexDG (monohexosyldiacylglycerol identified based on MGDG standards), DHexDG (dihexosyldiacylglycerol identified based on DGDG standards), Cer, Hex-cer, and Dihex-cer, of T. gondii and its host fibroblasts was performed, using electrospray ionization tandem mass spectrometry (ESI-MS/MS). As shown in , when the lipid classes were considered as mol% of the total of these lipids from each source, Toxoplasma gondii was found to be enriched in PC (57.4% vs. 43.4%), PE-cer (2.4% vs. 0.01%), and PA (0.7% vs. 0.5%) relative to its host fibroblasts, while the host fibroblasts had (as mol%) relatively more ePC, ePE, lysoPE, diacyl PS, SM, and Hex-cer.
FIGURE 1 Composition of lipid classes in host fibroblasts (A) and Toxoplasma gondii (B). Lipids are determined from the total of those lipid classes shown. Numbers above each column indicate the mol% value for that species. The detection of MHexDG and DHexDG was (more ...)
Glycerolipid molecular species
An advantage of ESI-MS/MS over traditional methodologies for lipid analysis is that individual lipid molecular species can be identified. In , PC molecular species from T. gondii and host fibroblasts are depicted. From these data, it is obvious that there are significant differences between the molecular species profile of the parasite and its host. Most strikingly, there are high levels of shorter-chain (28-, 30- and 32-carbons in the combined two acyl chains) species in the parasite lipids. PCs with a combined count of 28 and 30 carbons make up about 21% of the T. gondii PC molecular species, while these species make up only 3% of the host molecular species. The most prominent of these 28 and 30 carbon species were identified by product ion analysis as 1–16:0,2–14:0 PC, 1–16:1,2–14:0 PC, di14:0 PC, and 1–16:0,2–12:0 PC (), indicating that saturated 14-carbon and even 12-carbon acyl species are used in phospholipids considerably more frequently by this parasite than by its host. While there are some qualitative differences, the amounts of 34-, 36- and 38-carbon (combined diacyl count) PC molecular species relative to the total polar lipids are roughly similar between the parasite and host. In the longer chain species, the parasite has more PC 40:7, which was shown to be 1–18:1,2–22:6 PC (). Overall, the PC of T. gondii appears to contain PC similar to the host with additional 28-, 30-, and 32-carbon species. Thus, the shorter-chain and mostly saturated species are fairly specific to the parasite.
FIGURE 2 Diacyl phosphatidylcholine molecular species of host fibroblasts and Toxoplasma gondii. Species shown in A and B are indicated by the total number of acyl carbons: the total number of double bonds. Species are indicated as mol% of total lipids in classes (more ...)
Table 1 Identification of lipid molecular species in Toxoplasma gondii tachyzoites. Selected lipid molecular species that represented a larger fraction of Toxoplasma gondii’s lipids than of fibroblast lipids were subjected to product ion analysis in the (more ...)
PE () of T. gondii is quite similar to its host in molecular species composition, but, as in PC, there are more 28- 30- and 32-carbon PE species in the parasite than in the host. Twenty-eight- and 30-carbon species make up about 1.6% of the parasite PE, but only 0.1% of the host PE. PE 30:0 from T. gondii was identified as 1–16:0,2–14:0 PE ().
FIGURE 3 Diacyl phosphatidylethanolamine molecular species of host fibroblasts and Toxoplasma gondii. Species shown in A and B are indicated by the total number of acyl carbons: the total number of double bonds. Species are indicated as mol% of total lipids in (more ...)
Whereas PI makes up a similar fraction of the polar lipids of T. gondii and host fibroblasts (), the molecular species of PI () of T. gondii are less dominated by the PI 38:4 molecular species, which makes up 48% of host PI and 10% of parasite PI. PI 34:1 makes up a greater percentage of the T. gondii PI (50%) than of the host PI (2%). This species was determined by product ion analysis () to be PI containing 16:0 and 18:1 fatty acyl species.
FIGURE 4 Diacyl phosphatidylinositol molecular species of host fibroblasts and Toxoplasma gondii. Species shown in A and B are indicated by the total number of acyl carbons: the total number of double bonds. Species are indicated as mol% of total lipids in classes (more ...)
PS molecular species are shown in . In general, T. gondii and host PS are similar, but host PS 36:1 species is common, making up about 24% of the total PS. T. gondii has only about half the relative amount of PS found in the host and has a more equitable distribution of PS among its molecular species, with the highest species (PS 36:2) making up 15% of the PS species.
FIGURE 5 Diacyl phosphatidylserine molecular species of host fibroblasts and Toxoplasma gondii. Species are indicated by the total number of acyl carbons: the total number of double bonds. Species are indicated as mol% of total lipids in classes shown in (more ...)
Besides the PC species composition, the most notable difference between the polar lipid composition of T. gondii and that of its host fibroblasts is in the sphingolipids containing phosphate. shows a scan of the sphingosine-containing compounds (i.e., precursors of m/z 264 in positive ion mode) in host fibroblasts () and T. gondii (). Both parasite and host contain Cer, Hex-cer, and, Dihex-cer, each with a predominance of 16- and 24-carbon fatty-amide species. Indeed, the sphingolipid spectra were qualitatively quite similar except for signals for SM (e.g., SM 16:0 at m/z 703) which was higher in the host cells than in T. gondii, and the presence of a large peak at m/z 661 and a smaller one at m/z 689, which were present in the parasite, but undetectable or at the limit of detection in the host cells. The SM 16:0 peak and other SM peaks were also detectable in a positive ion scan for precursors of phosphocholine (Pre 184) and peaks at m/z 661 and m/z 689 were also present in the positive ion scan for the neutral loss of phosphoethanolamine (NL 141) (data not shown). Peaks at m/z 661 and m/z 689 were identified by product ion analysis as PE-cer 16:0 and PE-cer 18:0; product ion spectra in the positive ion mode are shown in . The structures of the SM 16:0, the predominant phosphosphingolipid of host fibroblasts, and PE-cer 16:0, the predominant phosphosphingolipid of T. gondii, are shown in . Sphingomyelin and PE-cer are related lipids with an ethanolamine moiety in PE-cer in contrast to a choline moiety in SM.
FIGURE 6 Sphingosine-containing molecular species of host fibroblasts and Toxoplasma gondii as shown by ESI MS/MS in the positive mode. (A and B) Precursors of m/z 264 (sphingosine) were identified as [M + H]+ ions. Ceramides, hexosyl ceramides, di-hexosyl ceramides, (more ...)
Structures of SM 16:0 and PE-cer 16:0 as [M + H]+ ions. (A) SM 16:0, the predominant phosphosphingolipid of host fibroblasts. (B) PE-cer 16:0, the predominant phosphosphingolipid of Toxoplasma gondii tachyzoites.
PE-cer and SM molecular species were quantified (). Because an authentic compound for PE-cer is not available, PE was quantified in relation to diacyl PE internal standards. Our lack of knowledge of a “response factor” for PE-cer only allows us to estimate the PE-cer content at about 2% of the polar lipids. Because the same method was used to analyze PE-cer in T. gondii and in the host cells, we were able to ascertain that T. gondii has a much higher, on the order of 100-fold higher, concentration of PE-cer than its host.
FIGURE 8 Quantification of sphingomyelin and phosphoethanolamine molecular species of host fibroblasts and Toxoplasma gondii. Species are indicated as mol% of total lipids in classes shown in and are indicated by the number of acyl carbons: the number (more ...)
Correction for host cell membrane contamination
In separate studies (data not shown), uninfected host cells were passed through a needle and a 3-micron filter in precisely the same manner as the infected cells. The lipidomic data obtained were subtracted from those obtained with the infected cells (i.e., those from the isolated parasites). Because the amount of filtered fibroblast lipids was less than 4% of that of the filtered T. gondii lipid species, employing this correction did not significantly change the lipid composition determined for the isolated parasites.