Identification and biochemical characterisation of Acanthamoeba castellanii cysteine protease 3
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(2020) 13:592 Wang et al. Parasites Vectors https://doi.org/10.1186/s13071-020-04474-8
Open Access
RESEARCH
Identification and biochemical characterisation of Acanthamoeba castellanii cysteine protease 3 Zhixin Wang1†, Duo Wu1†, Hiroshi Tachibana2, Meng Feng1* and Xun‑jia Cheng1*
Abstract Background: 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. Methods: 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 follow‑ ing coculture with AcCP3 gene knockdown trophozoites by Western blotting. Results: 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 tropho‑ zoites 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 signal‑ ling pathways in HCECs decreased after inhibiting the expression of trophozoite AcCP3. Conclusions: 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. Keywords: Acanthamoeba castellanii, Cysteine protease, Virulence factor, Encystment, p53 pathway
*Correspondence: [email protected]; [email protected] † Zhixin Wang and Duo Wu contributed equally to this work 1 Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China Full list of author information is available at the end
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