Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Front Cell Infect Microbiol. 2021 Mar 12;11:627888. doi: 10.3389/fcimb.2021.627888. eCollection 2021.
The surface molecule gp82 of metacyclic trypomastigote (MT) forms of , the protozoan parasite that causes Chagas disease, mediates the host cell invasion, a process critical for the establishment of infection. Gp82 is known to bind to the target cell in a receptor-dependent manner, triggering Ca signal, actin cytoskeleton rearrangement and lysosome spreading. The host cell receptor for gp82 was recently identified as LAMP2, the major lysosome membrane-associated protein. To further clarify the mechanisms of MT invasion, we aimed in this study at identifying the LAMP2 domain that interacts with gp82 and investigated whether target cell PKC and ERK1/2, previously suggested to be implicated in MT invasion, are activated by gp82. Interaction of MT, or the recombinant gp82 (r-gp82), with human epithelial HeLa cells induced the activation of Ca-dependent PKC and ERK1/2. The LAMP2 sequence predicted to bind gp82 was mapped and the synthetic peptide based on that sequence inhibited MT invasion, impaired the binding of r-gp82 to HeLa cells, and blocked the PKC and ERK1/2 activation induced by r-gp82. Treatment of HeLa cells with specific inhibitor of focal adhesion kinase resulted in inhibition of r-gp82-induced PKC and ERK1/2 activation, as well as in alteration of the actin cytoskeleton architecture. PKC activation by r-gp82 was also impaired by treatment of HeLa cells with inhibitor of phospholipase C, which mediates the production of diacylglycerol, which activates PKC, and inositol 1,4,5-triphosphate that releases Ca from intracellular stores. Taken together, our results indicate that recognition of MT gp82 by LAMP2 induces in the host cell the activation of phosholipase C, with generation of products that contribute for PKC activation and the downstream ERK1/2. This chain of events leads to the actin cytoskeleton disruption and lysosome spreading, promoting MT internalization.
环状动基体（MT）形式的锥虫表面分子 gp82 介导了宿主细胞的入侵，这是感染建立的关键过程。gp82 以受体依赖的方式与靶细胞结合，引发 Ca 信号、肌动蛋白细胞骨架重排和溶酶体扩散。gp82 的宿主细胞受体最近被鉴定为 LAMP2，即主要的溶酶体膜相关蛋白。为了进一步阐明 MT 入侵的机制，我们旨在本研究中确定与 gp82 相互作用的 LAMP2 结构域，并研究先前被认为与 MT 入侵有关的靶细胞 PKC 和 ERK1/2 是否被 gp82 激活。MT 或重组 gp82（r-gp82）与人类上皮 HeLa 细胞的相互作用诱导 Ca 依赖性 PKC 和 ERK1/2 的激活。预测与 gp82 结合的 LAMP2 序列被映射，基于该序列的合成肽抑制了 MT 的入侵，阻止了 r-gp82 与 HeLa 细胞的结合，并阻断了 r-gp82 诱导的 PKC 和 ERK1/2 的激活。用粘着斑激酶的特异性抑制剂处理 HeLa 细胞，导致 r-gp82 诱导的 PKC 和 ERK1/2 激活的抑制，以及肌动蛋白细胞骨架结构的改变。r-gp82 诱导的 PKC 激活也被用 PLC 抑制剂处理 HeLa 细胞所损害，PLC 介导二酰基甘油的产生，从而激活 PKC，以及肌醇 1,4,5-三磷酸从细胞内储存中释放 Ca。综上所述，我们的结果表明，MT gp82 被 LAMP2 识别，在宿主细胞中诱导 PLC 的激活，产生的产物有助于 PKC 的激活和下游 ERK1/2。这一连串的事件导致肌动蛋白细胞骨架的破坏和溶酶体的扩散，促进 MT 的内化。
