细菌病原体在细胞和有机水平宿主反应探针中的应用
Summary
我们描述了可用于分析主机编码因子活性的体外和体内感染检测。
Abstract
细菌病原体有多种策略, 一旦在真核细胞中存活和增殖。所谓的 “细胞增生李斯特菌” 病原体(单核细胞增生李斯特菌、志贺氏菌、假克氏菌、杜拉伦斯法郎和环结节) 可进入受感染的细胞溶胶。物理和酶降解的主要空泡膜。一旦进入细胞溶胶, 这些病原体既会增殖, 又会产生足够的机械力, 穿透宿主细胞的质膜, 从而感染新的细胞。在这里, 我们展示了 l . 单核细胞基因 (lm) 细胞感染周期的这一结束步骤如何通过成菌性系和流式细胞术来量化, 并举例说明病因和宿主编码因素是如何影响这一点的。过程。我们还显示了体外感染的培养细胞的 lm 感染动力学与体内感染小鼠的肝细胞的lm感染动力学的密切对应关系。这些基于功能的检测相对简单, 可以很容易地扩展到基于发现的高通量屏幕的真核细胞功能的调制器。
Introduction
基于感染的实验模型具有内在的挑战性, 因为它们依赖于宿主和病原体的起始状态条件、各种病原体感染策略以及难以归因病原和宿主驱动的过程基于结果。单核细胞增生李斯特菌(lm) 由于其遗传和微生物的可追踪性、快速和持续的细胞感染策略以及相对清晰的特性, 已成为一种理想的病原体, 可以探测宿主防御反应。其纤维素和有机水平的感染表型之间的关系。lm的细胞感染通过四个不同的阶段1: (i) 细胞入侵, 结束与lm被封闭在一个液泡内;(二) 以lm为导向溶解液泡膜, 并将lm 释放到细胞溶胶中;(iii) 组织内的复制;(iv) 质膜的物理渗透, 导致直接相邻的细胞 (如上皮细胞) 的感染, 或在单生细胞中, 将lm释放到细胞外环境中。每个阶段都是由特定的 lm编码因素 (称为 “毒力因素”) 推动的, 这些因素一旦被删除, 会导致细胞和动物模型的感染缺陷。这种一般的感染策略是由一些所谓的 “细胞质” 病原体2独立进化而来的。
菌落形成单元 (cfu) 检测被广泛用于评估体外 (即细胞) 和体内 (即有机) 感染结果。除了高灵敏度, 特别是对体内感染, cfu 检测提供了一个明确的读数病原体入侵和细胞内存活/增殖。cfu 检测已被广泛用于分析影响感染的lm和宿主细胞决定因素。尽管这些先前的研究提供了丰富的信息, 以分析细胞入侵和结肠内复制, cfu 的检测, 据我们所知, 还没有被用来跟踪第四阶段的lm感染过程: 细胞逃逸。在这里, 我们描述了相对简单的方法, 如何监测细胞逃逸 (以下简称 “出现”) 可以通过 cfu 分析 (以及流式细胞术), 并展示了如何病理和宿主编码的因素如何调节这个阶段的lm感染周期。对细胞lm感染周期的终末期进行分析, 可以确定更多的病原体和宿主细胞感染特有的因素和活动。
Protocol
根据所有适当的政府关于美国国立卫生研究院实验动物护理和使用的准则, 对老鼠进行了人道的治疗, 迈阿密大学动物护理机构批准了它们的使用和使用委员会 (议定书 16-053)。 1. 准备感染细胞 在 dmem 组织培养基中传播小鼠巨噬细胞样细胞系 raw 264.7, 辅以10% 的胎儿小牛血清 (以下简称 dmemce/fcs), 使用125ml 瓶和组织培养孵化器保持在 37°c% co2. 感染前一天, ?…
Representative Results
评估病原体和宿主编码因素对细胞感染的影响使用上述感染条件, 在与培养的巨噬细胞共同培养1.5 小时后, 将有0.15 的输入野生类型lm恢复 (图 1a)。在随后的1.5 小时共同孵化 (感染后 3小时, hpi) 中, 可行的lm的恢复增加了 4倍, 从 3 hpi 增加了4倍到 6 hpi, 存活可行的lm的恢复又增加了7.5倍。由于细胞不溶于细…
Discussion
由于其快速和持续的细胞感染计划, lm是一个理想的病原体, 探索细胞活动, 影响感染。已经确定了一些对lm细胞感染 11、12、13、14的积极或消极影响的宿主因素。在我们的实验室中表征的两个这样的宿主因子, 即 Perforin-2 和 heme 管制抑制剂 (p2 和 hi), 调节了 lm感染过程的显著特征。?…
Divulgations
The authors have nothing to disclose.
Materials
| ACK Lysing Buffer | Gibco | A1049201 | |
| ArC Amine Reactive Compensation Beads | Life Technologies | A10346 | |
| BHI (Brain Heart Infusion) broth | EMD Milipore | 110493 | |
| Cell Strainer, 70 µm | VWR | 10199-656 | |
| Collagenase D | Roche | 11088858001 | |
| DMEM media | Gibco | 11965-092 | |
| FACS tubes | BD Falcon | 352054 | |
| FBS – Heat Inactivated | Sigma-Aldrich | F4135-500ML | |
| Hanks’ Balanced Salt solution | Sigma-Aldrich | H6648-6X500ML | |
| LB agar | Grow Cells MS | MBPE-4040 | |
| LIVE/DEAD Fixable Yellow Dead Cell Stain kit | Life Technologies | L349S9 | |
| Rhodamine Phalloidin | Thermo Fischer | R415 | |
| SP6800 Spectral Analyzer | Sony | ||
| Syringe 28G 1/2" 1cc | BD | 329461 | |
| TPP Tissue Culture 48 Well Plates | MIDSCI | TP92048 | |
| TPP Tissue Culture 6 Well Plates | MIDSCI | TP92406 | |
| UltraComp eBeads | eBioscience | 01-2222-42 | |
| Antigen | |||
| CD11b | Biolegend | Flurochrome = PE Cy5, Dilution = 1/100, Clone = M1/70 | |
| CD11c | Biolegend | Flurochrome = AF 647, Dilution = 1/100, Clone = N418 | |
| CD45 | Biolegend | Flurochrome = APC Cy7, Dilution = 1/100, Clone = 30-F11 | |
| F4/80 | Biolegend | Flurochrome = PE, Dilution = 1/100, Clone = BM8 | |
| Live/Dead | Invitrogen | Flurochrome = AmCyN, Dilution = 1/100 | |
| Ly6C | Biolegend | Flurochrome = PacBlue, Dilution = 1/200, Clone = HK1.4 | |
| MHC II | Biolegend | Flurochrome = AF 700, Dilution = 1/200, Clone = M5/114.15.2 | |
| NK 1.1 | Biolegend | Flurochrome = BV 605, Dilution = 1/100, Clone = PK136 |
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Citer Cet Article
Gayle, P., Freitag, N. E., Strbo, N., Schesser, K. Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (144), e58775, doi:10.3791/58775 (2019).