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 equivocal with trace amounts of several species just at the limit of detection and of uncertain significance. Error bars are standard deviation (n = 4). The asterisks indicate lipid classes that differed significantly (p < 0.05) in mol% in Toxoplasma gondii as compared to the host fibroblast.

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 shown in Figure 1. Error bars are standard deviation (n = 4). (A) Diacyl phosphatidylcholine molecular species of host fibroblasts. (B) Diacyl phosphatidylcholine molecular species of Toxoplasma gondii. Species marked by the arrows, chosen because they are higher in Toxoplasma than in the host fibroblasts, were subjected to product ion analysis (Table 1).

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 classes shown in Figure 1. Error bars are standard deviation (n = 4). (A) Diacyl phosphatidylethanolamine molecular species of host fibroblasts. (B) Diacyl phosphatidylethanolamine molecular species of Toxoplasma gondii. The species marked by an arrow, chosen because it is higher in Toxoplasma than in the host fibroblasts, was subjected to product ion analysis (Table 1).

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 shown in Figure 1. Error bars are standard deviation (n = 4). (A) Diacyl phosphatidylinositol molecular species of host fibroblasts. (B) Diacyl phosphatidylinositol molecular species of Toxoplasma gondii. The species marked by an arrow, chosen because it is higher in Toxoplasma than in the host fibroblasts, was subjected to product ion analysis (Table 1).

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 Figure 1. Error bars are standard deviation (n = 4). (A) Diacyl phosphatidylserine molecular species of host fibroblasts. (B) Diacyl phosphatidylserine molecular species of Toxoplasma gondii.

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, and tri-hexosyl ceramides are identified in both host fibroblasts and Toxoplasma gondii as indicated with their amide-linked fatty acyl species. SM 16:0 is identified in host fibroblasts at m/z 703, but produced only a very small signal in Toxoplasma. It should be noted that SM produces m/z 264 only as a minor fragment, in contrast to other sphingolipids, which produce m/z 264 as a major fragment; thus the signal for SM under-represents its amount in relation to the amounts of the other sphingolipids. (A) Sphingosine-containing molecular species of host fibroblasts. (B) Sphingosine-containing molecular species of Toxoplasma gondii. In the Toxoplasma gondii extract, PE-cer produced signals at m/z 689 for the 18:0 amide-linked species and at m/z 661 for the 16:0 amide-linked species. Product ion analysis of these species is shown in C and D. These species were absent from the precursors of m/z 264 scan of the host fibroblast extract (A). (C and D) Product ion analysis of the Toxoplasma phosphatidylethanolmine molecular species, “PE-cer 18:0” (m/z 689) (C) and “PE-cer 16:0” (m/z 661) (D). The species indicated by the arrow in B were subjected, as an [M + H]⁺ ions (*), to product ion analysis to confirm their identifications. Note that these product ion scans were performed on an unfractionated Toxoplasma extract, and so isobaric species, i.e. species with the same nominal mass as the molecular ion of interest may produce fragment ions in addition to those derived from the ion of interest. The m/z 264 ion, indicated as “a”, is the characteristic ion for sphingosine (a dihydroxy 18-carbon sphingoid base). The ions indicated as “b”, m/z 308 in A and m/z 280 in B, are characteristic of the fatty amide species, 18:0 and 16:0, respectively. Fragmentation of SM 16:0 produced the same m/z 280 ion, characteristic of the fatty amide (22). The ions labeled “c” and “d” are produced by a neutral loss of phosphoethanolamine (NL 141) (“d”) and an additional water loss (“c”).

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.

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 Figure 1 and are indicated by the number of acyl carbons: the number of double bonds, assuming that the ceramide contains a dihydroxy18:1 base (sphingosine); this is demonstrated in Figure 6C and 6D for the species indicated. Error bars are standard deviation (n = 5). (A) Sphingomyelin and phosphoethanolamine molecular species of host fibroblasts. (B) Sphingomyelin and phosphoethanolamine molecular species of Toxoplasma gondii. The species marked by the arrows, chosen because they are higher in Toxoplasma than in the host fibroblasts, were subjected to product ion analysis.