Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Front Cell Infect Microbiol. 2021 Oct 19;11:769722. doi: 10.3389/fcimb.2021.769722. eCollection 2021.
Metacyclic trypomastigote (MT) forms of have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-β-cyclodextrin (MβCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MβCD, were incubated in serum-containing medium or in nutrient-depleted PBS, and the CM generated under these conditions was analyzed by Western blotting. In PBS, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MβCD-treated parasites. CM from untreated and MβCD-treated G strain, generated in PBS, inhibited CL strain internalization. Treatment of CL strain MTs with MβCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which releases surface molecules implicated in host cell invasion.
已证实，循环亲环体（MT）形式的会以囊泡或可溶性形式释放到调节宿主细胞入侵的阶段特异性表面分子 gp82 和 gp90 进入培养基中。在这里，我们发现，在侵袭性差的 G 株与宿主细胞相互作用期间，gp82 和 gp90 以囊泡样形式释放，而高度侵袭性 CL 株则没有观察到这种释放。通过扫描电子显微镜观察到 CL 和 G 株释放的大小不一的囊泡，两种菌株的条件培养基（CM）蛋白谱相似，但 gp82 和 gp90 的含量不同，Western 印迹中均在 G 株中检测到高强度的 gp82 和 gp90 条带，而在 CL 株中则几乎检测不到。共聚焦图像显示 CL 和 G 株 MT 表面 gp82 和 gp90 的分布明显不同。在细胞入侵实验中，添加 G 株 CM 可导致 CL 株内化减少。用特异性 mAb 偶联磁珠处理 G 株 CM 耗尽 gp82，可增加其对 CL 株入侵的抑制作用，而耗尽 gp90 则相反。还研究了胆固醇耗竭药物甲基-β-环糊精（MβCD）对 MT 释放 gp82 和 gp90 的影响。未处理或用 MβCD 处理的 G 株 MT 在含血清的培养基或营养缺乏的 PBS 中孵育，并通过 Western blot 分析在此条件下生成的 CM。在 PBS 中，未经处理的 MT 释放的 gp82 和 gp90 水平低于用 MβCD 处理的寄生虫。来自未处理和 MβCD 处理的 G 株的 CM，在 PBS 中生成，可抑制 CL 株的内化。用 MβCD 处理 CL 株 MT 可导致 gp82 和 gp90 脱落增加，宿主细胞入侵减少。还研究了 PLC 对 gp82 和 gp90 脱落的影响。与未处理的寄生虫相比，用特异性 PLC 抑制剂预处理的 G 株 MT 的 CM 中 gp82 和 gp90 的含量较低。我们的研究结果有助于阐明释放参与宿主细胞入侵的表面分子的机制。
