Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
Parasit Vectors. 2020 Nov 23;13(1):592. doi: 10.1186/s13071-020-04474-8.
Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs) belonging to the cathepsin L-family and explores the mechanism of AcCP3 interaction with host cells.
Six AcCP genes were amplified by polymerase chain reaction (PCR). Quantitative real-time PCR was used to analyse the relative mRNA expression of AcCPs during the encystation process and between pre- and post-reactivated trophozoites. To further verify the role of AcCP3 in these processes, AcCP3 recombinant proteins were expressed in Escherichia coli, and the hydrolytic activity of AcCP3 was determined. The influence of the AcCP3 on the hydrolytic activity of trophozoites and the toxicity of trophozoites to human corneal epithelial cells (HCECs) was examined by inhibiting AcCP3 expression using siRNA. Furthermore, the levels of p-Raf and p-Erk were examined in HCECs following coculture with AcCP3 gene knockdown trophozoites by Western blotting.
During encystation, five out of six AcCPs exhibited decreased expression, and only AcCP6 was substantially up-regulated at the mRNA level, indicating that most AcCPs were not directly correlated to encystation. Furthermore, six AcCPs exhibited increased expression level following trophozoite reactivation with HEp-2 cells, particularly AcCP3, indicating that these AcCPs might be virulent factors. After refolding of recombinant AcCP3 protein, the 27 kDa mature protein from the 34 kDa pro-protein hydrolysed host haemoglobin, collagen and albumin and showed high activity in an acidic environment. After AcCP3 knockdown, the hydrolytic activity of trophozoite crude protein against gelatin was decreased, suggesting that these trophozoites had decreased toxicity. Compared with untreated trophozoites or negative control siRNA-treated trophozoites, AcCP3-knockdown trophozoites were less able to penetrate and damage monolayers of HCECs. Western blot analysis showed that the activation levels of the Ras/Raf/Erk/p53 signalling pathways in HCECs decreased after inhibiting the expression of trophozoite AcCP3.
AcCP6 was correlated to encystation. Furthermore, AcCP3 was a virulent factor in trophozoites and participated in the activation of the Ras/Raf/Erk/p53 signalling pathways of host cells.
棘阿米巴属是自由生活的阿米巴原虫，广泛分布于环境中。本研究检测了属于组织蛋白酶 L 家族的致病棘阿米巴半胱氨酸蛋白酶（AcCP），并探讨了 AcCP3 与宿主细胞相互作用的机制。
通过聚合酶链反应（PCR）扩增了 6 个 AcCP 基因。采用定量实时 PCR 分析了 AcCPs 在包囊形成过程中和前、后再激活滋养体之间的相对 mRNA 表达。为了进一步验证 AcCP3 在这些过程中的作用，在大肠杆菌中表达 AcCP3 重组蛋白，并测定 AcCP3 的水解活性。通过 siRNA 抑制 AcCP3 的表达，研究 AcCP3 对滋养体水解活性和滋养体对人角膜上皮细胞（HCEC）毒性的影响。此外，通过 Western blot 检测与 AcCP3 基因敲低滋养体共培养后 HCEC 中 p-Raf 和 p-Erk 的水平。
在包囊形成过程中，6 个 AcCP 中有 5 个表达下调，只有 AcCP6 在 mRNA 水平上显著上调，表明大多数 AcCP 与包囊形成没有直接关系。此外，用 HEp-2 细胞再激活滋养体后，6 个 AcCP 的表达水平升高，特别是 AcCP3，表明这些 AcCP 可能是毒力因子。重组 AcCP3 蛋白复性后，34 kDa 的前蛋白水解为 27 kDa 的成熟蛋白，可水解宿主血红蛋白、胶原蛋白和白蛋白，并在酸性环境中表现出高活性。AcCP3 敲低后，滋养体粗蛋白对明胶的水解活性降低，提示这些滋养体毒性降低。与未经处理的滋养体或阴性对照 siRNA 处理的滋养体相比，AcCP3 敲低的滋养体穿透和破坏 HCEC 单层的能力降低。Western blot 分析显示，抑制滋养体 AcCP3 表达后，HCEC 中 Ras/Raf/Erk/p53 信号通路的激活水平降低。
AcCP6 与包囊形成有关。此外，AcCP3 是滋养体中的一种毒力因子，参与宿主细胞中 Ras/Raf/Erk/p53 信号通路的激活。
