Live In Vivo Imaging of Plasmodium Invasion of the Mosquito Midgut
Live In Vivo Imaging of Plasmodium Invasion of the Mosquito Midgut
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DOI：
10.1128/msphere.00692-20
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发表时间：
2020-09-01
期刊：
影响因子：
4.8
通讯作者：
Barillas-Mury, Carolina
中科院分区：
文献类型：
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作者：
Trisnadi, Nathanie;Barillas-Mury, Carolina
蚊子中肠是疟原虫必须克服的关键屏障，以完成其发育周期并传播到新的脊椎动物宿主。以前的共聚焦研究与固定感染中肠表明，动合子穿越中肠上皮细胞，并造成不可逆的组织损伤。在这里，我们调查的时空动态动合子中肠遍历和中肠细胞入侵的反应。建立了一种新的安装策略，确定了合适的荧光染料组合，并优化了在体内标记蚊子组织的方案，并开发了使用共聚焦显微镜的实时成像方案。跟踪数据显示，在中肠表面滑行的动合子比留在管腔中或侵入上皮的动合子移动得更快、更远。图像分析证实，寄生虫入侵和细胞穿越发生在几分钟内，而受损细胞中的半胱天冬酶活性，细胞凋亡的指示，和F-肌动蛋白细胞骨架重排的细胞挤出到肠腔持续数小时。这种时间差异突出了血细胞介导的细胞免疫和蚊子补体系统的重要性，以建立协调和有效的抗疟原虫反应。这种新的在体内成像协议使我们能够连续观察个别动合子活蚊子内的肠腔和细胞遍历过程中，并捕捉随后的细胞反应入侵在真实的时间为几个hours，而不损失的组织integrity.IMPORTANCE疟疾是人类最具破坏性的寄生虫病之一，是由按蚊蚊子传播。蚊子中肠是疟原虫必须克服的关键屏障，以完成其发育周期并传播到新的宿主。在这里，我们开发了一种新的策略来可视化疟原虫动合子，因为它们穿过蚊子中肠，并通过成像活蚊子来跟踪受损上皮细胞的反应。了解这些相互作用的空间和时间方面是至关重要的，当开发新的策略来破坏疾病的传播。
The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new vertebrate host. Previous confocal studies with fixed infected midguts showed that ookinetes traverse midgut epithelial cells and cause irreversible tissue damage. Here, we investigated the spatiotemporal dynamics of ookinete midgut traversal and the response of midgut cells to invasion. A novel mounting strategy was established, suitable fluorescent dye combinations were identified and protocols optimized to label mosquito tissues in vivo, and live imaging protocols using confocal microscopy were developed. Tracking data showed that ookinetes gliding on the midgut surface travel faster and farther than those that remain in the lumen or those that have invaded the epithelium. Image analysis confirmed that parasite invasion and cell traversal occur within a couple of minutes, while caspase activity in damaged cells, indicative of cellular apoptosis, and F-actin cytoskeletal rearrangements in cells extruded into the gut lumen persist for several hours. This temporal difference high-lights the importance of hemocyte-mediated cellular immunity and the mosquito complement system to mount a coordinated and effective antiplasmodial response. This novel in vivo imaging protocol allowed us to continuously observe individual ookinetes in live mosquitoes within the gut lumen and during cell traversal and to capture the subsequent cellular responses to invasion in real time for several hours, without loss of tissue integrity.IMPORTANCE Malaria is one of the most devastating parasitic diseases in humans and is transmitted by anopheline mosquitoes. The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new host. Here, we developed a new strategy to visualize Plasmodium ookinetes as they traverse the mosquito midgut and to follow the response of damaged epithelial cells by imaging live mosquitoes. Understanding the spatial and temporal aspects of these interactions is critical when developing novel strategies to disrupt disease transmission.