Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu 862, São Paulo, SP 04023-900, Brazil.
J Eukaryot Microbiol. 2013 Jul-Aug;60(4):377-87. doi: 10.1111/jeu.12043. Epub 2013 May 9.
We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log-phase promastigotes treated with 1 μM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 μM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 μM myriocin-treated parasites, respectively. High percentage of myriocin-treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 μM myriocin, respectively). Transmission electron microscopy following treatment with 1 μM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3-ketodihidrosphingosine to myriocin-treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid-free mutant, where addition of ethanolamine rescue wild-type parasite characteristics.
我们研究了丝氨酸棕榈酰转移酶抑制剂——美罗霉素，对培养的巴西利什曼原虫（Viannia）前鞭毛体的影响。美罗霉素可显著减少磷脂酰肌醇磷酸神经酰胺的合成，这是前鞭毛体中表达的主要鞘脂，通过薄层色谱和电喷雾电离质谱来表征。对数生长期前鞭毛体用 1μM 美罗霉素处理后，生长速度降低了 52%，形态发生改变，如形状更圆，鞭毛更短。用美罗霉素处理的前鞭毛体也显示出多种异常的细胞表型。用 1 或 5μM 美罗霉素处理后，具有一个核和一个动基体（1N1K）的细胞比例分别从 89%（对照值）降至 27%或 3%。具有两个核（2N2K）的细胞比例分别从 7%（对照值）增加到 19%和 6%，分别为 1 或 5μM 美罗霉素处理的寄生虫。用美罗霉素处理的寄生虫中，高比例的寄生虫表现为具有三个或更多核的大型非典型细胞（分别为 32%和 89%，对于 1 或 5μM 美罗霉素）。用 1μM 美罗霉素处理后，通过透射电子显微镜观察到存在 4N 寄生虫，可能是不完全有丝分裂的结果。在美罗霉素处理的前鞭毛体中添加 3-酮二氢鞘氨醇可挽救寄生虫的生长和形态。添加乙醇胺不能挽救美罗霉素对寄生虫的影响。我们的研究结果表明，鞘脂对完成有丝分裂至关重要，并且可能在巴西利什曼原虫的细胞增殖中发挥主要作用，因此与描述的无鞘脂的利什曼原虫游离突变体的数据不同，在添加乙醇胺后，野生型寄生虫的特征得到了挽救。
