Denny Paul W, Goulding David, Ferguson Michael A J, Smith Deborah F
Wellcome Trust Laboratories for Molecular Parasitology, Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK.
Mol Microbiol. 2004 Apr;52(2):313-27. doi: 10.1111/j.1365-2958.2003.03975.x.
Sphingolipids are structural components of the eukaryotic plasma membrane that are involved, together with cholesterol, in the formation of lipid microdomains (rafts). Additionally, sphingolipid metabolites have been shown to modulate a wide variety of cellular events, including differentiation and apoptosis. To investigate the role of de novo sphingolipid biosynthesis in Leishmania, we have focused on serine palmitoyltransferase (SPT), which catalyses the first, rate-limiting step in the synthetic pathway. Genetic ablation of one SPT subunit, LmLCB2, yields viable null parasites that can no longer synthesize ceramide and sphingolipids de novo. Unexpectedly, LmLCB2 expression (and sphingolipid biosynthesis) is stage regulated in Leishmania, being undetectable in intramacrophage parasites. As expected from this observation, the LmLCB2 null mutants maintain infectivity in vivo. However, they are compromised in their ability to form infective extracellular parasites, correlating with a defect in association of the virulence factor, leishmanolysin or GP63, with lipid rafts during exocytosis and an observed relocalization of a second virulence factor, lipophosphogycan, during differentiation. Thus, de novo sphingolipid biosynthesis is critical for membrane trafficking events in extracellular Leishmania but has at best a minor role in intracellular pathogenesis.
鞘脂是真核细胞质膜的结构成分，与胆固醇一起参与脂质微区（脂筏）的形成。此外，鞘脂代谢产物已被证明可调节多种细胞活动，包括分化和凋亡。为了研究从头合成鞘脂在利什曼原虫中的作用，我们聚焦于丝氨酸棕榈酰转移酶（SPT），它催化合成途径中的第一步且是限速步骤。对一个SPT亚基LmLCB2进行基因敲除，产生了可存活的缺失型寄生虫，这些寄生虫无法再从头合成神经酰胺和鞘脂。出乎意料的是，LmLCB2的表达（以及鞘脂的生物合成）在利什曼原虫中受阶段调控，在巨噬细胞内的寄生虫中无法检测到。基于这一观察结果，正如预期的那样，LmLCB2缺失型突变体在体内保持感染性。然而，它们形成感染性细胞外寄生虫的能力受损，这与毒力因子利什曼溶素或GP63在胞吐过程中与脂筏的结合缺陷以及在分化过程中观察到的第二个毒力因子脂磷酸聚糖的重新定位有关。因此，从头合成鞘脂对于细胞外利什曼原虫中的膜转运事件至关重要，但在细胞内发病机制中充其量只起次要作用。
