利用利什曼原虫破译毛孔形成毒素的分子机制和功能
Summary
这里介绍的是使用 利什曼原虫主要 前鞭毛体来确定孔形成毒素诱导的结合、细胞毒性和信号传导的方案。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 杆菌 中可用的遗传突变体来确定毒素抗性的新机制。
Abstract
了解造孔毒素（PFT）的功能和机制具有挑战性，因为细胞可以抵抗PFT引起的膜损伤。虽然生物物理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全部补充的还原论方法。培养的人类细胞提供了一种替代系统，但它们的复杂性和修复机制的冗余使得识别特定机制变得困难。相比之下，导致皮肤利什曼病的人类原生动物病原体，即大利什曼原虫，在复杂性和生理相关性之间提供了最佳平衡。杆菌在遗传上是可处理的，可以在体外培养到高密度，并且可以在已建立的小鼠模型中测量扰动对感染的任何影响。此外，L. major合成的脂质与哺乳动物不同，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佳特征毒素家族胆固醇依赖性溶细胞素（CDC）的PFT进行探测。CDC与利什曼原虫膜中的麦角甾醇结合，可杀死大鞭毛乳杆菌，表明杆菌是确定PFT功能的细胞和分子机制的合适模型系统。这项工作描述了在L. majorpromagotes中测试PFT功能的方法，包括寄生虫培养，用于评估脂质敏感性的遗传工具，膜结合测定和细胞死亡测定。这些测定将能够快速使用L. major作为强大的模型系统，用于了解一系列进化上多样化的生物体的PFT功能和脂质组织的共性。
Introduction
孔形成毒素（PFTs）是细菌毒素中最大的家族1，但它们穿孔和破坏细胞的机制知之甚少。研究得最好的毛孔形成毒素家族是胆固醇依赖性溶细胞素（CDCs）。CDC主要由革兰氏阳性菌合成，包括坏死性筋膜炎的病原体化 脓性链球菌2。 化脓性 链球菌分泌CDC链球菌溶血素O（SLO），其作为单体与宿主细胞质膜中的甾醇结合，低聚化并将~20-30nm孔插入膜1。脂质在这一过程中所起的作用仍然不确定。
研究脂质-CDC相互作用的一种方法是使用化学定义的脂质体。虽然定义的脂质体提供了维持毒素结合和孔形成所需的脂质阈值的信息3，4，但它们并不能完全概括细胞功能。例如，重组脂质体缺乏哺乳动物宿主的脂质不对称性和响应毒素的脂质修饰5。脂质体的一种替代方案是使用哺乳动物细胞系。虽然这些细胞系在生理上更具相关性，但在毒素传感和抗性机制方面存在很大程度的冗余2。因此，用于抵抗CDC的修复途径仍然确定得很差。值得注意的是，Ca2+的流入是膜修复1的主要激活剂。在Ca2+涌入的下游，有多个途径参与，包括神经酰胺依赖性修复6，7和MEK依赖性修复途径6。这些途径与其他蛋白质效应物相互作用，包括运输所需的内体分选复合物（ESCRT）8和膜联蛋白6，9，10。由于冗余，在哺乳动物细胞中解剖这些途径具有挑战性，这混淆了数据解释。
在剖析修复途径的复杂性与简单性之间取得平衡的一种方法是使用更简单的生物，例如利什曼原虫属中的原生动物病原体。利什曼原虫在人类和其他动物中引起利什曼病。利什曼病的范围从皮肤利什曼病（自限性皮肤病变）到致命的内脏利什曼病（肝脾肿大），取决于物种和其他因素11。重型利什曼原虫是皮肤利什曼病的病原体，通过白蛉媒介传播给人类，用于了解利什曼原虫的功能和感染12。此外，利什曼原虫属是12。它们作为细胞内哺乳动物巨噬细胞寄生虫存在，称为无鞭毛虫，并在白蛉中作为自由游泳的鞭毛原鞭毛体存在12。主要前鞭毛乳杆菌可以在补充血清的培养基（如M199）中培养至高密度13。前乳鞭毛在遗传上也是可处理的;存在许多基因敲除，包括那些靶向脂质生物合成途径的基因敲除13。这些敲除可以通过感染Balb / c小鼠13来评估感染性和病变发展的生长和差异。
除了利什曼原虫培养的相对容易和脂质生物合成敲除的范围外，寄生虫的基因组比哺乳动物更简单。利什曼原虫最具特征的物种是L. major，它具有许多现有的遗传工具，例如脂质代谢缺陷的突变体14。值得注意的是，许多修复蛋白缺失。迄今为止，杆菌尚未发现关键哺乳动物修复蛋白（如膜联蛋白）的同系物。这使得能够表征进化上保守的修复途径，而没有哺乳动物系统的复杂性。然而，迄今为止，利什曼原虫尚未发现修复途径的特征。同时，参与修复的关键信号通路，如MEK通路6，在利什曼原虫sp.15，16中是保守的，尽管同系物需要验证。丝裂原活化蛋白激酶（MAPK）途径在墨西哥乳杆菌中得到了很好的研究，它有助于哺乳动物细胞的细胞内存活和热稳定性，并控制变环发生16。在利什曼原虫属中，15 种 MAPK 中有 10 种被表征为17.根据保守磷酸化唇序列中的身份，预测LmMAPK9和LmMAPK13与哺乳动物ERK1 / 2最相似。磷酸化唇序列对于哺乳动物ERK1 / 2和LmMAPK9和LmMAPK13都是TEY。然而，其中八个利什曼原虫MAPK具有TDY磷酸化基序15。在利什曼原虫、LmxMKK18 和 MEKK 相关激酶 （MRK1）19 中至少发现了两种 MEK 同系物。这表明在利什曼原虫中发现的见解可以转化为哺乳动物系统。在它们不能转化为哺乳动物系统的情况下，它们代表了治疗利什曼病的治疗靶点。
为了使用 L. majorpromasgotes 来研究膜修复和与毒素的相互作用，需要中等通量技术。虽然高分辨率活细胞成像能够实时可视化标记的蛋白质和膜，但它的通量很低，可能无法测量细胞存活率。中等通量活性测定包括通过流式细胞术测量染料摄取、线粒体活性测量或乳酸脱氢酶 （LDH） 等细胞蛋白的释放。在哺乳动物细胞中，LDH测定不能定量测量细胞死亡20。此外，基于群体的测定（如LDH释放或线粒体活性）不允许进行稳健的单细胞或多参数分析20。相比之下，基于流式细胞术的测定可实现多参数单细胞分析20。然而，这些测定尚未应用于了解毒素生物学或 对主要前鞭毛乳杆菌 毒素的反应。
在这项研究中，SLO被用作了解两种不同缓冲液中重 乳杆菌 鞘脂零突变体的质膜扰动的工具 – 通常用于培养 杆菌 的M199培养基和更简单的Tyrode缓冲液。描述了中等通量流式细胞术测定并用于生成毒素剂量-反应曲线。将流式细胞仪检测数据建模为逻辑曲线，以确定LC50 值。利用这些信息，可以确定SLO的亚溶解剂量，以便使用蛋白质印迹法验证MAPK抗体。
Protocol
Representative Results
Discussion
本研究以人类病原虫大利 什曼原虫 为模型系统，描述了肺功能转移疗法分子机制和功能的研究方法。开发了一种基于中通量流式细胞术的细胞毒性测定法来测量单细胞活力。活力在人群水平上是定量的，因为LC50 值可以使用逻辑建模从剂量-反应曲线计算出来。作为原理验证，使用流式细胞术测定来说明培养基的选择可以改变野生型和鞘脂缺陷的 L.major 对SLO的敏感性，并确定…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者要感谢Keyel和Zhang实验室的成员对手稿的批判性审查。作者感谢艺术与科学学院显微镜学院使用设施。
Materials
| 1.2 mL microtiter (Marsh) tubes | Fisher | 02-681-376 | Cytotoxicity assay |
| 1.5 mL microcentrifuge tube | Fisher | 05-408-129 | Toxin dilutions |
| 15 mL centrifuge tube | Avantor VWR (Radnor, PA) | 89039-666 | To hold cells and media |
| 1x Phosphate buffered saline (PBS) | Fisher | BP399 | For cell processing |
| 3% H2O2 | Walmart (Fayetteville, AR) | N/A | For ECL |
| 5x M199 | Cell-gro | 11150067 | Basal growth media for L. major promastigotes |
| Biosafety cabinet | Baker | To culture cells in sterile conditions | |
| Bovine serum albumin (BSA) | Fisher | BP1605-100 | Fraction V acceptable purity |
| CaCl2 | Fisher | BP510-100 | Stock concentration 100 mM |
| Centrifuge | Thermo Fisher | Heraeus Megafuge 40R | To pellet the cells from culture |
| Cy5 Mono-reactive dye pack | Cytiva (Marlborough, MA) | PA25031 | Fluorophore label for toxins |
| Digital dry bath | Benchmark | BSH1002 | To denature protein samples |
| EGTA | Amresco | 0732-100G | Stock concentration 0.5 M |
| Excel | Microsoft (Redmond, VA) | Data analysis software | |
| Flow cytometer (4-laser Attune NxT) | Fisher | Cytometer for data acquisition | |
| FlowJo | BD (Ashland, OR) | Software | |
| Formaldehyde | Fisher | BP531-500 | Fixative for counting cells |
| G418 | Fisher | BP673-1 | Selection agent for cells |
| Hellmanex III | Sigma | Z805939 | Dilute 1:4 for cleaning cytometer |
| Hemacytometer | Fisher | 0267151B | For counting cells |
| Human red blood cells | Zen-bio (Durham, NC) | SER-10MLRBC | To validate toxin activity |
| Ice bucket | |||
| Light microscope | Nikon | Eclipse 55i | To visualize cells |
| Nitrocellulose | Fisher | 88018 | For probing proteins via antibodies |
| Pipettors and tips | Avantor VWR | To dispense reagents | |
| Power supply | Bio-Rad | To run SDS-PAGE and transfers | |
| Propidium iodide | Biotium | 40016 | Stock concentration 2 mg/mL in water |
| Protein ladder | Bio-Rad | 161-0373 | To determine molecular weight of proteins |
| SDS-PAGE Running Apparatus (Mini Protean III) | Bio-Rad | 165-3302 | To separate proteins based on their size |
| Sealing tape | R&D | DY992 | To seal plates with cells |
| Streptolysin O C530A plasmid insert | Cloned into pBAD-gIII vector (Reference: 7) | ||
| Streptolysin O C530A toxin | Lab purified | Specific activity 4.34 x 105 HU/mg | |
| Swinging bucket rotor | Thermo Fisher | 75003607 | To centrifuge cells |
| V-bottom plate | Greiner Bio-one | 651206 | For cytotoxicity assay |
| Vortex | Benchmark | BV1000 | To mix cells |
| Western blot imaging system (Chemi-doc) | Bio-Rad | To visualize proteins by western blot | |
| Western Blot Transfer Apparatus (Mini Protean III) | Bio-Rad | 170-3930 | Transfer proteins to nitrocellulose |
| Whatman Filter paper | GE Healthcare Life Sciences | 3030-700 | Used in transfer of proteins to nitrocellulose |
| Antibody | |||
| Anti-ERK antibody | Cell Signaling Technologies | Cat# 9102S | Rabbit (1:1000 dilution) |
| Anti-lipophosphoglycan (LPG) antibody | CreativeBioLabs | Cat# WIC79.3 | Mouse (1: 1000) |
| Anti-MEK antibody | Cell Signaling Technologies | Cat# 9122L | Rabbit (1:1000) |
| Anti-mouse IgG, HRP conjugate | Jackson Immunoresearch | Cat#715-035-151 | Donkey (1:10000) |
| Anti-phosphoERK antibody | Cell Signaling Technologies | Cat# 9101S | Rabbit (1:1000) |
| Anti-pMEK antibody | Cell Signaling Technologies | Cat# 9121S | Rabbit (1:1000) |
| Anti-rabbit IgG, HRP conjugate | Jackson Immunoresearch | Cat#711-035-152 | Donkey (1:10000) |
| Anti-tubulin antibody | Sigma | Cat# T5168 | Mouse (1: 2000) |
| Leishmania major Genotypes | Reference: 13 | ||
| Episomal addback (spt2–/+SPT2) | Δspt2::HYG/Δspt2:PAC/+pXG-SPT2 | ||
| Serine palmitoyltransferase subunit 2 knockout (spt2–) | Δspt2::HYG/Δspt2::PAC | ||
| Wild type (WT) | LV39 clone 5 (Rho/SU/59/P) |
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