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首页> 外文学位 >Flagellar membrane targeting, immune evasion, and protein palmitoylation in the protozoan parasite Trypanosoma brucei.
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Flagellar membrane targeting, immune evasion, and protein palmitoylation in the protozoan parasite Trypanosoma brucei.

机译：原生动物寄生虫布鲁氏锥虫中的鞭毛膜靶向，免疫逃逸和蛋白质棕榈酰化。

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摘要

Trypanosomes are unicellular eukaryotic parasites responsible for tremendous morbidity and mortality worldwide. The research described herein focuses on Trypanosoma brucei, the etiologic agent of African sleeping sickness and a model organism for the related Trypanosoma cruzi and Leishmania spp., which cause Chagas disease and the leishmaniases, respectively. Trypanosomes express a family of myristoylated and palmitoylated calcium-binding proteins, the calflagins in T. brucei, which specifically localize to the flagellar membrane. To define the molecular determinants of calflagin localization, point mutations were introduced into the calflagin amino terminus. Assessment of point mutant acylation state and localization demonstrated the sufficiency of myristoylation for plasma membrane localization, and the requirement of secondary palmitoylation for specific sorting to the flagellar membrane. Contemporaneous with calflagin flagellar targeting upon palmitoylation was an association with lipid raft microdomains, which are enriched in the flagellar membrane. To identify and localize the enzyme responsible for calflagin palmitoylation and trafficking, a genome-wide screen for likely palmitoyl acyltransferases (PATs) was performed, followed by generation of knock-down mutants for each of twelve candidates. Testing each of these mutants led to the identification of the single enzyme mediating calflagin palmitoylation, TbPAT7. Calflagin palmitoylation by TbPAT7 is the first PAT-substrate pair identified in a protozoan organism and the first palmitoyl acyltransferase in any system shown to confer flagellar targeting. Surprisingly, TbPAT7, like its substrates, was found to localize to the flagellar membrane. These findings support a model, likely applicable to all ciliated eukaryotic cells, wherein enzyme-specific palmitoylation at the flagellar membrane favors the retention of protein substrates through interactions with the unique lipid composition of this organelle.;To determine the biologic function of the calflagins, calflagin deficient mutants were engineered and subjected to extensive phenotypic analysis. Despite being unaffected in terms of their in vitro viability, growth, motility, and morphology, the virulence of these mutants was greatly attenuated in a murine model of infection. Compared to parental wild type cells, which proliferated unchecked to cause 100% host mortality within 10 days post-infection, calflagin mutants demonstrated a normal initial rise in parasitemia, followed by clearance to undetectable levels within 8 days post-infection. Outgrowth of parasite populations was associated with antigenic variation of the VSG surface coat, indicating that the initial population had been cleared in an antigen-specific manner. A mechanism for the increased susceptibility of calflagin mutants to the early host response was suggested by a relevant in vitro experiment. When surface-labeled with anti-VSG antibodies, calflagin mutants showed delayed endocytosis and degradation of these antibodies, a deficit that is expected to render parasites susceptible to host humoral immunity. Moreover, the localization of the calflagins was shown to be critical for their function, since inhibition of TbPAT7, which causes a redistribution of calflagins to the pellicular rather than flagellar membrane, likewise prolonged host survival and suppressed parasitemias during infection. These finding implicate the calflagins as important virulence factors through their contribution to evasion of host immunity.;With a specific role established for palmitoylation in the flagellar trafficking of calflagins, examination of protein palmitoylation was then extended to the global level, first by characterization of mutants for each of the twelve T. brucei PATs, and then by identification of the total cellular palmitoyl proteome. Despite the lethality of chemical pan-PAT inhibition, no single enzyme appears essential to parasite growth, indicating either enzymatic compensation or non-canonical PAT activity for those substrates essential to parasite proliferation. Acyl-biotin exchange and streptavidin chromatography enabled the purification and identification of cellular T. brucei palmitoyl proteins, including 3 of 4 previously described palmitoyl proteins and another 49 novel palmitoyl proteins. The identities of these proteins implicate palmitoylation in broad aspects of trypanosome biology, including parasite metabolism, subcellular trafficking, and cell signaling. Unexpectedly, the identification of certain novel palmitoyl proteins furthermore offers an explanation for previously enigmatic processes, including the chain length specificity of fatty acyl CoA synthetases and the dynamic regulation of folate/pterin transporters.;Taken together, the research described in this thesis represents a substantial and novel contribution to the field of trypanosome biology, identifying a novel pathway for flagellar/ciliary trafficking, elucidating an important role for calcium-signaling proteins in immune evasion and pathogenesis, and providing the first global analysis of the enzyme mediators and protein substrates of palmitoylation in these pathogenic parasites.
机译：锥虫是单细胞的真核寄生虫，在世界范围内引起巨大的发病率和死亡率。本文所述的研究集中于非洲昏睡病的病原体布鲁氏锥虫和相关的克鲁斯锥虫和利什曼原虫属的模型生物，它们分别引起恰加斯病和利什曼原虫病。锥虫表达了一个家族化的肉豆蔻酰化和棕榈酰化的钙结合蛋白，布氏锥虫中的钙鞭毛蛋白，特异性地定位在鞭毛膜上。为了定义钙鞭蛋白定位的分子决定因素，将点突变引入钙鞭蛋白氨基末端。对点突变体酰化状态和定位的评估表明，肉豆蔻酰化足以实现质膜定位，并且需要次级棕榈酰化才能对鞭毛膜进行特异性分选。与钙鞭毛鞭毛同时靶向棕榈酰化的过程是与脂筏微结构域相关的，脂筏微结构域富含鞭毛膜。为了鉴定和定位负责钙鞭蛋白棕榈酰化和运输的酶，对可能的棕榈酰酰基转移酶（PAT）进行了全基因组筛选，然后生成了十二种候选基因的敲低突变体。测试这些突变体中的每一个导致鉴定介导钙鞭蛋白棕榈酰化的单一酶TbPAT7。由TbPAT7产生的钙鞭毛蛋白棕榈酰化是原生动物中鉴定出的第一个PAT底物对，并且在显示可赋予鞭毛靶向的任何系统中，都是第一个棕榈酰酰基转移酶。出乎意料的是，发现TbPAT7与其底物一样位于鞭毛膜上。这些发现支持了一种可能适用于所有纤毛真核细胞的模型，其中鞭毛膜上的酶特异性棕榈酰化作用通过与该细胞器独特脂质成分的相互作用而有利于蛋白质底物的保留。为了确定钙鞭毛蛋白的生物学功能，对钙鞭毛蛋白缺陷型突变体进行了工程改造，并进行了广泛的表型分析。尽管在体外存活力，生长，运动性和形态方面不受影响，但是在小鼠感染模型中这些突变体的毒力大大减弱了。与亲本野生型细胞相比，亲本野生型细胞在感染后10天内未经检查而增殖，导致100％的宿主死亡，而钙鞭毛蛋白突变体显示出正常的初始寄生虫病升高，随后在感染后8天内清除至检测不到的水平。寄生虫种群的生长与VSG表面涂层的抗原变异有关，表明初始种群已以抗原特异性方式清除。一项相关的体外实验表明，钙鞭蛋白突变体对早期宿主反应的敏感性增加的机制。当用抗VSG抗体进行表面标记时，钙鞭毛蛋白突变体显示出这些抗体的内吞作用延迟和降解，这一缺陷有望使寄生虫易感宿主免疫。此外，由于对TbPAT7的抑制作用会导致钙鞭毛蛋白重新分布到防护膜而不是鞭毛膜上，因此，钙鞭毛蛋白的定位对它们的功能至关重要，同样可以延长宿主的存活时间并抑制感染期间的寄生虫病。这些发现表明钙鞭毛蛋白通过逃避宿主免疫而成为重要的毒力因子。通过在棕榈鞭毛的鞭毛运输中确定棕榈酰化的特殊作用，蛋白质棕榈酰化的检测随后扩展到了全球水平，首先是通过突变体的表征对于十二种布鲁氏杆菌PATS中的每一种，然后通过鉴定总的细胞棕榈酰蛋白质组来确定。尽管化学泛PAT抑制具有致命性，但没有任何一种酶似乎对寄生虫生长必不可少，这表明对寄生虫增殖必不可少的那些底物进行酶促补偿或非经典PAT活性。酰基生物素交换和链霉亲和素色谱法能够纯化和鉴定细胞布鲁氏杆菌棕榈酰蛋白，包括先前描述的4种棕榈酰蛋白中的3种和另外49种新型棕榈酰蛋白。这些蛋白质的身份在锥虫生物学的广泛方面暗示了棕榈酰化作用，包括寄生虫代谢，亚细胞运输和细胞信号传导。出乎意料的是，某些新型棕榈酰蛋白的鉴定进一步解释了先前的神秘过程，包括脂肪酰基辅酶A合成酶的链长特异性和叶酸/蝶呤转运蛋白的动态调节。对锥虫生物学领域的重大创新贡献，确定鞭毛/纤毛贩运的新途径，阐明钙信号蛋白在免疫逃逸和发病机理中的重要作用，并首次对这些病原体中棕榈酰化的酶介体和蛋白底物进行了全面的分析。

著录项

  • 作者

    Emmer, Brian T.;

  • 作者单位

    Northwestern University.;

  • 授予单位 Northwestern University.;
  • 学科 Biology Cell.;Biology Parasitology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 193 p.
  • 总页数 193
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:26

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