Inositol phosphorlyceramide synthase null Leishmania major are viable and virulent in animal infections where salvage of host sphingomyelin predominates

Abstract

Many pathogens synthesize inositolphosphorylceramide (IPC) as the major sphingolipid (SL), unlike the mammalian host where sphingomyelin or more complex SLs predominate. Thus, divergence between IPCS and mammalian sphingolipid synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, promastigotes lacking de novo sphingolipid synthesis (∆spt2-) survive and remain virulent, as amastigotes salvage host sphingolipids and are still able to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in L. major (Δipcs-). Unexpectedly and unlike fungi where IPCS is essential, Δipcs- was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC itself were absent in Δipcs- promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian sphingomyelin was highly abundant in purified amastigotes from both WT and Δipcs-. Quantitative estimation showed SM to be about 5-fold more abundant than IPC in WT, suggesting that normally SM is the dominant amastigote SL. Analysis of the WT amastigote IPC ceramide anchor showed that it arose not from de novo synthesis by the parasite, but through catabolism of mammalian SLs, probably through the action of the parasite sphingomyelinase ISCL. These data suggest that SM salvage may account for the survival and virulence of Δipcs-, a finding which likely mitigates the suitability of IPCS as a chemotherapeutic target.

Sphingolipids protect ergosterol in theLeishmania majormembrane from sterol-specific toxins

“…Elevation of ceramide levels and lack of IPC were confirmed by mass spectrometry (Kuhlmann et al, 2022). Wild type L. major LV39, spt2 -, ipcs -, smtand isclwere cultured at 27°C in M199 medium with 0.182% NaHCO 3 , 40 mM Hepes, pH 7.4, 0.1 M adenine, 1 µg/mL biotin, 5 µg/mL hemin & 2 µg/mL biopterin and 10% heat inactivated fetal bovine serum (FBS), pH 7.4.…”

“…In mammalian cells, phospholipids regulate cholesterol access [33]. We did not examine amastigotes because they salvage lipids from the host macrophage [34][35][36], the membranes are substantially similar in spt2 -, ipcs -, and wild type amastigotes [20,37], and contaminating macrophage membranes could obscure results.…”

“…In mammalian cells, phospholipids regulate cholesterol access (Lange et al , 2013). We did not examine amastigotes because they salvage lipids from the host macrophage (Henriques et al , 2003; Moitra et al , 2021; Winter et al , 1994), the membranes are substantially similar in spt2 — , ipcs — , and wild type amastigotes (Kuhlmann et al , 2022; Zhang et al , 2005), and contaminating macrophage membranes could obscure results. Since S. pyogenes contributes to secondary bacterial infections during cutaneous leishmaniasis (Gimblet et al , 2017; Jayasena Kaluarachchi et al , 2021), future work is needed to determine if CDCs contribute to late stage infection, and/or the time required for resolution.…”

“…described. The inositol phosphorylceramide synthase knockout Δipcs::HYG/Δipcs::PAC (ipcs -) and episomal addback Δipcs::HYG/Δipcs::PAC /+pXG-IPCS (ipcs -/+IPCS) were generated in the Beverley lab [20]. Elevation of ceramide levels and lack of IPC were confirmed by mass spectrometry [20].…”

“…Δsmt::HYG/Δsmt::PAC/+pXG-SMT (smt -/+SMT) (Mukherjee et al, 2019), sterol 14-αdemethylase knockout Δc14dm:HYG/Δc14dm::PAC (c14dm -) (Mukherjee et al, 2020), and episomal addback Δc14dm:HYG/Δc14dm::PAC/+pXG-C14DM (c14dm -/+C14DM) (Mukherjee et al, 2020) have been previously described. The inositol phosphorylceramide synthase knockout Δipcs::HYG/Δipcs::PAC (ipcs -) and episomal addback Δipcs::HYG/Δipcs::PAC /+pXG-IPCS (ipcs -/+IPCS) were generated in the Beverley lab (Kuhlmann et al, 2022).…”