Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins using Leishmania major.
Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins using Leishmania major.
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DOI：
10.3791/64341
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发表时间：
2022-10-28
期刊：
影响因子：
--
通讯作者：
Keyel PA
中科院分区：
文献类型：
--
作者：
Haram CS;Moitra S;Keane R;Breslav E;Zhang K;Keyel PA
了解孔形成毒素（PFT）的功能和机制是具有挑战性的，因为细胞抵抗PFT引起的膜损伤。虽然生物物理学方法有助于理解孔的形成，但它们通常依赖于缺乏膜脂质和蛋白质的完整补充的还原论方法。培养的人类细胞提供了一种替代系统，但它们在修复机制上的复杂性和冗余性使得识别特定机制变得困难。相比之下，负责皮肤利什曼病的人类原生动物病原体，利什曼原虫，提供了复杂性和生理相关性之间的最佳平衡。L. Major是遗传上易处理的，并且可以在体外培养至高密度，并且可以在建立的鼠模型中测量扰动对感染的任何影响。此外，L. major合成不同于哺乳动物的脂质，这可能会改变膜动力学。膜动力学的这些改变可以用来自最具特征的毒素家族胆固醇依赖性溶细胞素（CDC）的PFT来探测。CDC与利什曼原虫膜上的麦角固醇结合，可以杀死利什曼原虫。major promastigotes，表明L. major是用于确定PFT功能的细胞和分子机制的合适模型系统。本文介绍了L.主要前鞭毛体，包括寄生虫培养，用于评估脂质易感性的遗传工具，膜结合试验和细胞死亡试验。这些测定将使L.主要作为一个强大的模型系统，了解PFT功能在一系列进化上不同的生物体和共性的脂质组织。这里提出的是一个协议，使用利什曼原虫主要前鞭毛体，以确定结合，细胞毒性，和信号诱导的孔形成毒素。提供了链球菌溶血素O的概念验证。其他毒素也可用于利用L.主要用于确定毒素抗性的新机制。
Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major, offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization. Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.
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影响因子：
3.1
作者：
Limbago, B;Penumalli, V;Scott, JR
通讯作者：
Scott, JR
影响因子：
--
作者：
Wiese, M;Kuhn, D;Grünfelder, CG
通讯作者：
Grünfelder, CG
DOI：
10.1016/j.bbamcr.2014.09.005
发表时间：
2015-09-01
影响因子：
5.1
作者：
Wolfmeier, Heidi;Schoenauer, Roman;Babiychuk, Eduard B.
通讯作者：
Babiychuk, Eduard B.
影响因子：
5.4
作者：
Zhang K
通讯作者：
Zhang K
DOI：
10.1155/2011/971968
发表时间：
2011
期刊：
Journal of signal transduction
影响因子：
--
作者：
Brumlik MJ;Pandeswara S;Ludwig SM;Murthy K;Curiel TJ
通讯作者：
Curiel TJ