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Sphingolipid synthesis and metabolism as a target for anti Giardial therapy.

机译：鞘脂的合成和代谢作为抗贾第虫治疗的目标。

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摘要

Giardia lamblia, a parasitic protozoan and a major cause of waterborne enteric disease worldwide, exists in two morphologic forms: (1) the infective trophozoite, and (2) the transmissible water-resistant cyst. Exposure of cysts to gastric acid during passage through the human stomach triggers excystation, while factors in the small intestine, where trophozoites colonize, induce encystation or cyst formation. Successful operation of this excystation-encystation cycle is important for this pathogen to survive, multiply, and differentiate in the human small intestine.; Sphingolipids and their metabolites have been shown to modulate a wide variety of cellular events, including apoptosis, cell signaling, and synthesis of membrane rafts. More recently, 3-keto-sphinganine, synthesized by serine-palmitoyl transferase (SPT) as the first product of sphingolipid biosynthesis, was shown to be involved in endocytosis in yeast. Interestingly, however, Giardia has a very limited ability to synthesize sphingolipids de novo and depends on exogenous sources for energy production and other metabolic activities. I hypothesize that sphingolipids scavenged by this parasite from its micro-environment or synthesized de novo (although this is limited) play key roles in regulating the life cycle of Giardia. To test my hypothesis, I used radioactive and fluorescent sphingolipid probes and various metabolic/pathway inhibitors to study the mechanism of sphingolipid internalization and targeting. Studies indicated that Giardia trophozoites have the ability to internalize and target intracellular ceramide, the major sphingolipid precursor, through clathrin- and cytoskeleton-based endocytic pathways. I speculated that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide and sphigomyelin molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.; In the second phase of my investigation, I used molecular and bioinformatic approaches to analyze sphingolipid metabolic genes in Giardia. Analyses revealed that within all sphingolipid biosynthesis pathways only five gene transcripts---serine-palmitoyltransferase 1 & 2 (spt-1 and 2), ceramide-glucosyltransferase (glcT-1), and sphingomyelinase B & 3b (smase b and 3b)---are differentially expressed in trophozoites and encysting cells. The spt-1 and spt-2 genes are expressed predominately in trophozoites, while glcT-1 and smase mRNAs are increased in encysting cells. Phylogenic and protein-family (Pfam) databases have indicated that genes for both SPT subunits (i.e., spt-1 and 2) in Giardia are closely related to prokaryotic amino acid transferases. Giardial glcT-1, on the other hand, belongs to the glucosyltransferase family 2 that is distributed widely in prokaryotes and eukaryotes. To test whether these genes and gene products are important for the growth and encystation of this waterborne pathogen, I used L-cycloserine (SPT inhibitor) and two newly available GlcT-1 inhibitors--- D-threo-1-phenyl-2-decanoyl-3-morpholinopropanol (PDMP) and D-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP)---on trophozoite and encysting cells. The results suggested that the inhibition of SPT by L-cycloserine induced morphological alterations and inhibited the endocytosis and intracellular targeting of fluorescently labeled ceramide dramatically, suggesting that the product of SPT enzyme (i.e., 3-keto-sphinganine) is in fact regulated the endocytosis in Giardia, as in Saccharomyces cerevisiae. On the other hand, PDMP and PPMP, two inhibitors of GlcT-1, reduced the production of vitro-derived cysts and damaged the cyst wall structures, as evidenced by DIC/confocal microscopy. These studies suggest that sphingolipids are important for giardial growth and encystation and that they could serve as potential targets for developing new therapies against Giardia and related mucosal parasites.
机译：贾第鞭毛虫是一种寄生虫，是世界范围内水源性肠道疾病的主要病因，以两种形态学形式存在：（1）滋养滋养体和（2）可传播的耐水囊肿。在通过人胃的过程中，将囊肿暴露于胃酸会触发兴奋，而滋养体定居的小肠中的因素会导致入侵或囊肿形成。兴奋-融合周期的成功运行对于这种病原体在人小肠中的生存，繁殖和分化至关重要。鞘脂及其代谢产物已显示出可调节多种细胞事件，包括细胞凋亡，细胞信号传导和膜筏的合成。最近，由丝氨酸-棕榈酰转移酶（SPT）合成的3-酮-鞘氨醇是鞘脂生物合成的第一个产物，被证明与酵母的胞吞作用有关。然而，有趣的是，贾第鞭毛虫具有从头合成鞘脂的能力非常有限，并且依赖外源来产生能量和进行其他代谢活动。我假设这种寄生虫从其微环境或从头合成（尽管这是有限的）清除的鞘脂在调节贾第鞭毛虫的生命周期中起关键作用。为了验证我的假设，我使用了放射性和荧光鞘脂探针以及各种代谢/途径抑制剂来研究鞘脂内在化和靶向作用的机制。研究表明，贾第鞭毛虫滋养体具有通过网格蛋白和细胞骨架的内吞途径内化和靶向细胞内神经酰胺（主要的鞘脂前体）的能力。我推测这种寄生的原生动物已经进化出了细胞骨架和网格蛋白依赖性的内吞机制，可以从细胞外部导入神经酰胺和蛇绿素分子，从而在生长和分化过程中合成膜和囊泡。在研究的第二阶段，我使用分子和生物信息学方法分析了贾第鞭毛虫的鞘脂代谢基因。分析显示，在所有鞘脂生物合成途径中，只有五个基因转录物-丝氨酸-棕榈酰转移酶1和2（spt-1和2），神经酰胺-葡萄糖基转移酶（glcT-1）和鞘磷脂酶B和3b（smase b和3b）- -在滋养体和吞噬细胞中差异表达。 spt-1和spt-2基因主要在滋养体中表达，而glcT-1和smase mRNA在进入细胞中增加。系统发育和蛋白质家族（Pfam）数据库表明，贾第鞭毛虫中两个SPT亚基的基因（即spt-1和2）与原核氨基酸转移酶密切相关。另一方面，贾第虫glcT-1属于在原核生物和真核生物中广泛分布的葡糖基转移酶家族2。为了测试这些基因和基因产物对于这种水生病原体的生长和侵染是否重要，我使用了L-环丝氨酸（SPT抑制剂）和两种新的GlcT-1抑制剂-D-threo-1-phenyl-2-滋养细胞和吞噬细胞上的癸酰基-3-吗啉代丙醇（PDMP）和D-苏--1-苯基-2-棕榈酰氨基-3-吗啉代-1-丙醇（PPMP）。结果表明，L-环丝氨酸对SPT的抑制可引起形态学改变，并显着抑制荧光标记的神经酰胺的内吞作用和细胞内靶向作用，表明SPT酶的产物（即3-酮-鞘氨醇）实际上在调节内吞作用。在贾第虫病中，就像酿酒酵母一样。另一方面，如DIC / Conconal显微镜检查所证实的，PDMP和PPMP是GlcT-1的两种抑制剂，减少了体外衍生的囊肿的产生并破坏了囊壁的结构。这些研究表明鞘脂对于贾第鞭毛虫的生长和侵入很重要，它们可以作为开发针对贾第鞭毛虫和相关粘膜寄生虫的新疗法的潜在靶标。

著录项

  • 作者

    Hernandez, Yunuen.;

  • 作者单位

    The University of Texas at El Paso.$bBiological Sciences.;

  • 授予单位 The University of Texas at El Paso.$bBiological Sciences.;
  • 学科 Biology Microbiology.; Biology Parasitology.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 106 p.
  • 总页数 106
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 微生物学;
  • 关键词

  • 入库时间 2022-08-17 11:39:08

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