Endocytosis and Sphingolipid Scavenging in Leishmania mexicana Amastigotes

Abstract

Leishmania species are the causative agents of the leishmaniases, a spectrum of neglected tropical diseases. Amastigote stage parasites exist within macrophages and scavenge host factors for survival, for example, Leishmania species utilise host sphingolipid for synthesis of complex sphingolipid. In this study L. mexicana endocytosis was shown to be significantly upregulated in amastigotes, indicating that sphingolipid scavenging may be enhanced. However, inhibition of host sphingolipid biosynthesis had no significant effect on amastigote proliferation within a macrophage cell line. In addition, infection itself did not directly influence host biosynthesis. Notably, in contrast to L. major, L. mexicana amastigotes are indicated to possess a complete biosynthetic pathway suggesting that scavenged sphingolipids may be nonessential for proliferation. This suggested that Old and New World species differ in their interactions with the macrophage host. This will need to be considered when targeting the Leishmania sphingolipid biosynthetic pathway with novel therapeutics.

Figure 1

The endocytosis of Texan Red labelled dextran at 32°C measured as described and with the background (uptake in control parasites at 0°C) subtracted. Results from a representative experiment, in triplicate with standard deviation shown. AFU: arbitrary fluorescence units.

Figure 2

HPTLC analysis of axenic procyclic promastigotes (aP) and amastigotes (aA) metabolically labelled with BODIPY FL C₅-ceramide. IPC: inositol phosphorylceramide; Cer: ceramide.

Figure 3

Invasion, % infected macrophages, determined 48 hours after infection in the presence or absence of the SPT inhibitor myriocin and with (FBS-DMEM) or without (FBS-reduced DMEM) exogenous serum in the media. Results of three independent experiments.

Figure 4

Proliferation, parasites per infected macrophage, determined 48 hours post infection in the presence or absence of the SPT inhibitor myriocin, and with (FBS-DMEM) or without (FBS-reduced DMEM) exogenous serum in the media. Results of three independent experiments.

Figure 5

Lysates of 5 × 10⁶ axenic procyclic promastigotes (aP) and amastigotes (aA) probed with the cross reacting LmjLCB2 and LmjNMT antibodies in a Western blot. Molecular weight markers in kDa shown on left of image.

Figure 6

HPTLC analysis of axenic amastigotes (aA) metabolically labelled with tritiated serine (Ser; 1.25 × 10⁷ cell equivalent). Axenic promastigotes (aP) similarly labelled with tritiated inositol (Inos; 5 × 10⁶ cell equivalent) served as markers. Following fluorography the plate was exposed to film for 15 days. PIP: phosphatidylinositol phosphate; IPC: inositol phosphorylceramide; PI: phosphatidylinositol; Cer: ceramide, migrating at solvent front.