Developmentally Regulated Sphingolipid Degradation in Leishmania major

Abstract

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl−) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl− mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl− promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl−. These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.

Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence

“…Recent studies have highlighted the essentiality of ISCL, a developmentally regulated neutral sphingomyelinase, in L. major virulence [10, 12]. Based on these reports, it may be of interest to characterize other enzymes involved in the degradation of phospholipids synthesized by the host or Leishmania .…”

“…Importantly, these ISCL-null mutants were hypersensitive to acidic pH [11] and lacked virulence in mice [10]. Infectivity was restored when ISCL-null mutants were complemented with a functional sphingomyelinase [10, 12]. These findings indicate that the turnover of host sphingolipids is essential for Leishmania survival in mammals.…”

Sphingolipid Degradation in Leishmania (Leishmania) amazonensis

“…L. amazonensis axenic amastigotes were cultured at 33°C in Grace's insect cell culture medium (the pH was adjusted to 5.3) with L-glutamine and 20% fetal bovine serum [28], [29]. Purification of lesion amastigotes were performed as previously described [10].…”

“…Importantly, iscl − mutants fail to cause pathology in either immunocompetent or immunodeficient mice [9]. Virulence of iscl − can be fully restored when a functional neutral sphingomyelinase (SMase) is introduced into these mutants [8], [10]. Consistent with its role as a virulence determinant, ISCL is preferentially expressed in the infective stages of L. major , i.e.…”

“…Consistent with its role as a virulence determinant, ISCL is preferentially expressed in the infective stages of L. major , i.e. stationary phase promastigotes and amastigotes [10]. The mechanism by which ISCL contributes to virulence is not well understood.…”