Identification and biochemical characterisation of Acanthamoeba castellanii cysteine protease 3

Abstract Background Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases ( Ac CPs) belonging to the cathepsin L-family and explores the mechanism of Ac CP3 interaction with host cells. Methods Six Ac CP genes were amplified by polymerase chain reaction (PCR). Quantitative real-time PCR was used to analyse the relative mRNA expression of Ac CPs during the encystation process and between pre- and post-reactivated trophozoites. To further verify the role of Ac CP3 in these processes, Ac CP 3 recombinant proteins were expressed in Escherichia coli , and the hydrolytic activity of Ac CP 3 was determined. The influence of the Ac CP3 on the hydrolytic activity of trophozoites and the toxicity of trophozoites to human corneal epithelial cells (HCECs) was examined by inhibiting Ac CP3 expression using siRNA. Furthermore, the levels of p-Raf and p-Erk were examined in HCECs following coculture with Ac CP3 gene knockdown trophozoites by Western blotting. Results During encystation, five out of six Ac CPs exhibited decreased expression, and only Ac CP 6 was substantially up-regulated at the mRNA level, indicating that most Ac CPs were not directly correlated to encystation. Furthermore, six Ac CPs exhibited increased expression level following trophozoite reactivation with HEp-2 cells, particularly Ac CP3, indicating that these Ac CPs might be virulent factors. After refolding of recombinant Ac CP3 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 Ac CP3 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, Ac CP3-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 Ac CP3. Conclusions Ac CP6 was correlated to encystation. Furthermore, Ac CP3 was a virulent factor in trophozoites and participated in the activation of the Ras/Raf/Erk/p53 signalling pathways of host cells. Graphical Abstract