秀 丽隐杆线虫 感染模型中的遗传免疫研究
Summary
秀丽隐杆线虫感染秀丽隐杆线虫感染使蠕虫能够产生对相同病原体具有高度抗性的后代。这是遗传免疫的一个例子，遗传免疫是一种鲜为人知的表观遗传现象。本方案描述了在遗传可处理的蠕虫模型中遗传免疫的研究。
Abstract
遗传免疫力描述了一些动物如何将先前感染的“记忆”传递给它们的后代。这可以提高其后代的病原体耐药性并促进生存。虽然许多无脊椎动物中已经报道了遗传免疫，但这种表观遗传现象背后的机制在很大程度上是未知的。天然微孢子病原体线虫皮病菌感染秀丽隐杆线虫感染，导致蠕虫产生的后代对微孢子虫具有很强的抵抗力。本方案描述了在简单且遗传可处理的巴黎猪笼草-秀丽隐杆线虫感染模型中的代际免疫研究。当前的文章描述了感染秀丽隐杆线虫和产生免疫启动后代的方法。还给出了通过微孢子虫染色和显微镜观察感染来测定对微孢子虫感染的抗性的方法。特别是，遗传免疫可防止宿主细胞被微孢子虫侵袭，荧光原位杂交（FISH）可用于量化侵袭事件。免疫引物后代中产生的微孢子孢子的相对量可以通过用甲壳素结合染料染色孢子来量化。迄今为止，这些方法已经阐明了遗传免疫的动力学和病原体特异性，以及其背后的分子机制。这些技术，加上可用于秀丽隐杆线虫研究的广泛工具，将使遗传免疫领域的重要发现成为可能。
Introduction
遗传免疫是一种表观遗传现象，父母暴露于病原体可以产生抗感染的后代。这种类型的免疫记忆已经在许多缺乏适应性免疫系统的无脊椎动物中得到证明，并且可以预防病毒，细菌和真菌疾病1。虽然遗传免疫对理解健康和进化具有重要意义，但这种保护背后的分子机制在很大程度上是未知的。这部分是因为许多描述遗传免疫力的动物不是用于研究的模型生物。相比之下，透明线虫 秀丽隐杆线虫 的研究受益于广泛的遗传和生化工具包2，3，高度注释的基因组4，5和短的生成时间。事实上， 对秀丽隐杆线虫 的研究已经在表观遗传学和先天免疫6，7领域取得了根本性的进步，现在它是研究免疫记忆的既定模型8，9。
微孢子虫是真菌病原体，可感染几乎所有动物，并在免疫功能低下的人中引起致命感染10。当微孢子孢子使用称为极性管的结构将其细胞内容物（孢子质）注入或“发射”到宿主细胞中时，感染就开始了。寄生虫的细胞内复制导致梅龙的形成，其最终分化成可以离开细胞的成熟孢子11，12。虽然这些寄生虫对人类健康和粮食安全都有害，但关于它们的感染生物学还有很多需要了解的地方12。线虫是一种天然的微孢子虫寄生虫，仅在蠕虫的肠细胞中复制，导致繁殖力降低，最终导致死亡。秀丽隐杆线虫感染模型已被用于显示：（1）自噬在病原体清除中的作用13，（2）微孢子虫如何非溶解地离开感染细胞14，（3）病原体如何通过形成合胞体15从细胞传播到细胞，（4）巴黎猪笼草用于与其宿主16接口的蛋白质，以及（5）转录细胞内病原体反应（IPR）的调节17，18.
秀丽隐杆线虫感染的方案在当前的工作中进行了描述，可用于揭示独特的微孢子虫生物学并剖析宿主对感染的反应。用甲壳素结合染料Direct Yellow 96（DY96）染色的固定蠕虫的显微镜检查显示含甲壳素的微孢子孢子在整个肠道中的感染扩散。DY96染色还可以可视化含甲壳素的蠕虫胚胎，以便同时评估蠕虫的重力（产生胚胎的能力），作为宿主适应性的读数。
最近的研究表明，感染了巴黎猪笼草的秀丽隐杆线虫产生的后代对相同的感染具有很强的抵抗力19。这种遗传性免疫持续一代并且是剂量依赖性的，因为来自感染更重的父母的后代对微孢子虫的抵抗力更强。有趣的是，巴黎猪笼草的后代对细菌性肠道病原体铜绿假单胞菌也更具抗性，尽管它们没有受到天然病原体奥赛病毒19的保护。本研究还表明，免疫引物的后代限制了微孢子虫对宿主细胞的侵袭。该方法还描述了免疫引物后代的收集以及如何使用FISH来检测肠细胞中的N. parisii RNA以测定宿主细胞入侵和孢子放电20。
总之，这些方案为研究 秀丽隐杆线虫的微孢子虫和遗传免疫提供了坚实的基础。希望该模型系统的未来工作能够在遗传免疫的新兴领域取得重要发现。这些技术也可能是研究微孢子虫诱导的其他宿主生物体遗传免疫的起点。
Protocol
Representative Results
Discussion
本方案描述了在简单 且遗传可 处理的秀丽隐杆线虫-秀丽隐杆线虫 感染模型中研究微孢子虫和遗传免疫。
孢子制备是一种密集的方案，通常产生足够的孢子进行6个月的实验，具体取决于生产率24。重要的是，在将其用于实验之前，必须确定每个新孢子“批次”的传染性。由于孢子制剂之间的传染性各不相同，因此必须一致地使用单个批次进行?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Winnie Zhao和Yin Chen Wan对稿件提供有益的评论。这项工作得到了加拿大自然科学和工程研究委员会（Grant #522691522691）的支持。
Materials
| 2.0 mm zirconia beads | Biospec Products Inc. | 11079124ZX | |
| 10 mL syringe | Fisher Scientific | 1482613 | |
| 5 μm filter | Millipore Sigma | SLSV025LS | |
| Axio Imager 2 | Zeiss | – | Fluorescent microscope for imaging of DY96- and FISH- stained worms on microscope slides |
| Axio Zoom V.16 Fluorescence Stereo Zoom Microscope | Zeiss | – | For live imaging of fluorescent transgenic animals to visualize the IPR |
| Baked EdgeGARD Horizontal Flow Clean Bench | Baker | – | |
| Bead disruptor, Genie SI-D238 Analog Disruptor Genie Cell Disruptor, 120 V | Global Industrial | T9FB893150 | |
| Cell-VU slide, Millennium Sciences Disposable Sperm Count Cytometers | Fisher Scientific | DRM600 | |
| Direct Yellow 96 | Sigma-Aldrich | S472409-1G | |
| EverBrite Mounting Medium with DAPI | Biotium | 23001 | |
| EverBrite Mounting Medium without DAPI | Biotium | 23002 | |
| Fiji/ImageJ software | ImageJ | https://imagej.net/software/fiji/downloads | |
| Mechanical rotor | Thermo Sceintific | 415110 / 1834090806873 | Used to spin tubes of bleached embryos for overnight hatching |
| MicroB FISH probe | Biosearch Technologies Inc. | – | Synthesized with a Quasar 570 (Cy3) 5' modification and HPLC purified, CTCTCGGCACTCCTTCCTG |
| N2 | Wild-type, Bristol strain | Default strain | Caenorhabditis Genetics Center (CGC) |
| Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | L3771-100G | |
| Sodium hydroxide solution (5 N) | Fisher Chemical | FLSS256500 | |
| Sodium hypochlorite solution (6%) | Fisher Chemical | SS290-1 | |
| Stemi 508 Stereo Microscope | Zeiss | – | For daily maintenance of worms and counting of L1 worms for assay set ups |
| Tween-20 | Sigma-Aldrich | P1379-100ML | |
| Vectashield + A16 | Biolynx | VECTH1500 |
References
- Tetreau, G., Dhinaut, J., Gourbal, B., Moret, Y. Trans-generational immune priming in invertebrates: current knowledge and future prospects. Frontiers in Immunology. 10, 1938 (2019).
- Au, V., et al. CRISPR/Cas9 methodology for the generation of knockout deletions in Caenorhabditis elegans. G3 Genes|Genomes|Genetics. 9 (1), 135-144 (2019).
- Kamath, R. Genome-wide RNAi screening in Caenorhabditis elegans. Methods. 30 (4), 313-321 (2003).
- The C. elegans Sequencing Consortium. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science. 282 (5396), 2012-2018 (1998).
- Yoshimura, J., et al. Recompleting the Caenorhabditis elegans genome. Genome Research. 29, 1009-1022 (2019).
- Weinhouse, C., Truong, L., Meyer, J. N., Allard, P. Caenorhabditis elegans as an emerging model system in environmental epigenetics: C. elegans as an environmental epigenetics model. Environmental and Molecular Mutagenesis. 59 (7), 560-575 (2018).
- Ermolaeva, M. A., Schumacher, B. Insights from the worm: the C. elegans model for innate immunity. Seminars in Immunology. 26 (4), 303-309 (2014).
- Willis, A. R., Sukhdeo, R., Reinke, A. W. Remembering your enemies: mechanisms of within-generation and multigenerational immune priming in Caenorhabditis elegans. TheFEBS Journal. 288 (6), 1759-1770 (2020).
- Burton, N. O., et al. Cysteine synthases CYSL-1 and CYSL-2 mediate C. elegans heritable adaptation to P. vranovensis infection. Nature Communications. 11, 1741 (2020).
- Wadi, L., Reinke, A. W. Evolution of microsporidia: an extremely successful group of eukaryotic intracellular parasites. PLoS Pathogens. 16, 1008276 (2020).
- Han, B., Takvorian, P. M., Weiss, L. M. Invasion of host cells by microsporidia. Frontiers in Microbiology. 11, 172 (2020).
- Tamim El Jarkass, H., Reinke, A. W. The ins and outs of host-microsporidia interactions during invasion, proliferation and exit. Cellular Microbiology. 22 (11), 13247 (2020).
- Balla, K. M., Lažetić, V., Troemel, E. R. Natural variation in the roles of C. elegans autophagy components during microsporidia infection. PLoS ONE. 14, 0216011 (2019).
- Szumowski, S. C., Estes, K. A., Troemel, E. R. Preparing a discreet escape: Microsporidia reorganize host cytoskeleton prior to non-lytic exit from C. elegans intestinal cells. Worm. 1 (4), 207-211 (2012).
- Balla, K. M., Luallen, R. J., Bakowski, M. A., Troemel, E. R. Cell-to-cell spread of microsporidia causes Caenorhabditis elegans organs to form syncytia. Nature Microbiology. 1 (11), 1-6 (2016).
- Reinke, A. W., Balla, K. M., Bennett, E. J., Troemel, E. R. Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins. Nature Communications. 8, 14023 (2017).
- Bakowski, M. A., et al. Ubiquitin-mediated response to microsporidia and virus infection in C. elegans. PLoS Pathogen. 10, 1004200 (2014).
- Reddy, K. C., et al. An intracellular pathogen response pathway promotes proteostasis in C. elegans. Current Biology. 27 (22), 3544-3553 (2017).
- Willis, A. R., et al. A parental transcriptional response to microsporidia infection induces inherited immunity in offspring. Science Advances. 7 (19), (2021).
- Tamim El Jarkass, H., et al. An intestinally secreted host factor promotes microsporidia invasion of C. elegans. eLife. 11, 72458 (2022).
- Solis, G. M., Petrascheck, M. Measuring Caenorhabditis elegans life span in 96 well microtiter plates. Journal of Visualized Experiments. 49, 2496 (2011).
- Stiernagle, T. Maintenance of C. elegans. WormBook. , (2006).
- Sutphin, G. L., Kaeberlein, M. Measuring Caenorhabditis elegans life span on solid media. Journal of Visualized Experiments. (27), e1152 (2009).
- Estes, K. A., Szumowski, S. C., Troemel, E. R. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells. PLOS Pathogens. 7, 1002227 (2011).
- Botts, M. R., Cohen, L. B., Probert, C. S., Wu, F., Troemel, E. R. Microsporidia intracellular development relies on myc interaction network transcription factors in the host. G3 Genes|Genomes|Genetics. 6 (9), 2707-2716 (2016).
- Corsi, A. K. A Transparent window into biology: A primer on Caenorhabditis elegans. WormBook. , 1-31 (2015).
- Rivera, D. E., Lažetić, V., Troemel, E. R., Luallen, R. J. RNA fluorescence in situ hybridization (FISH) to visualize microbial colonization and infection in the Caenorhabditis elegans intestines. bioRxiv. , (2022).
- Zhang, G., et al. A large collection of novel nematode-infecting microsporidia and their diverse interactions with Caenorhabditis elegans and other related nematodes. PLoS Pathogens. 12, 1006093 (2016).
- Luallen, R. J., et al. Discovery of a natural microsporidian pathogen with a broad tissue tropism in Caenorhabditis elegans. PLoS Pathogens. 12, 1005724 (2016).
- Troemel, E. R., Félix, M. -. A., Whiteman, N. K., Barrière, A., Ausubel, F. M. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans. PLoS Biology. 6, 309 (2008).
- Burton, N. O., et al. Intergenerational adaptations to stress are evolutionarily conserved, stress-specific, and have deleterious trade-offs. eLife. 10, 73425 (2021).
- Jaroenlak, P., et al. 3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery. PLoS Pathogens. 16, 1008738 (2020).
- Weidner, E., Manale, S. B., Halonen, S. K., Lynn, J. W. Protein-membrane interaction is essential to normal assembly of the microsporidian spore invasion tube. The Biological Bulletin. 188 (2), 128-135 (1995).
