A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Abstract

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

Keywords: Naegleria fowleri; cyst; cysteine protease inhibitor; encystation.

Figure 1

Multiple sequence alignment and phylogenetic analysis. (A) Multiple sequence alignment. The deduced amino acid sequence of N. fowleri (NfCPI) was aligned with the sequences of related proteins derived from other protozoa and humans. The Gln-Val-Val-Ala-Gly (QVVAG) cystatin motif is presented as a bold red line on the sequences. The predicted N-terminal signal peptide sequences are underlined. The predicted putative N-glycosylation sites in NfCPI are marked by asterisks. A potential disulfide bridge is presented in brackets and the associated cysteine residues are boxed in red color. The shading displays the degree of identity among the sequences. Sequence identity between NfCPI and other related proteins is shown on the right side. Sequence identity between NfCPI and other related proteins is shown on the right side. Naegleria gruberi CPI (XP_002680758.1), Fowlerstefin (NF0067710), Acanthamoeba castellanii CPI (AET79741), Dictyostelium discoideum CPI (XP_629960), Human cystatin A (NP_005204), Human cystatin B (NM_000100), Human cystatin C (NP_000090), and Human cystatin D (NP_001891) are included in the alignment. (B) The phylogenetic tree was constructed by the maximum likelihood method using the MEGA6 program. Yellow, cystatin A and B clades; blue, cystatin C, D, and S clades; pink, CPIs from plants. Numbers on the branches show bootstrap proportion (1000 replicates).

Figure 2

Expression of recombinant NfCPI and structural analysis. (A) The NfCPI was purified using Ni–NTA affinity chromatography and analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1, non-induced E. coli lysate; lane 2, isopropyl-1-thio-β-d-galactopyranoside (IPTG)-induced E. coli lysate; lane 3, Ni–NTA affinity purified NfCPI. (B) The NfCPI was mixed with different concentrations of SDS (0–10%, v/v) and β-ME with or without heating and analyzed via SDS-PAGE. (C) Gel filtration chromatography analysis. The NfCPI was loaded to Superdex 200 HR 10/30 column and fractions (0.5 mL) were collected. The collected fractions were analyzed by SDS-PAGE and their inhibitory activities against NfCB and NfCBL were assayed. The fractions expressing strong inhibitory activity (fractions 26 to 28) are represented as red bars and confirmed by SDS-PAGE. The K_av value of NfCPI (red dot) was calculated by comparing with those of size markers (black dots).

Figure 3

Inhibitory activity of NfCPI. (A) Inhibitory activity of NfCPI against cysteine proteases. Different concentrations of NfCPI (0–100 nM) were incubated with each cysteine protease (10 nM) and the residual enzyme activity of each enzyme was assayed. (B) pH dependency. NfCPI was incubated with papain, NfCB and NfCBL in different pH buffers and the residual enzyme activity was analyzed. (C) pH stability. NfCPI was incubated in different pH buffers for 3 h and its inhibitory activity against cysteine proteases was assayed. (D) Thermal stability. NfCPI was incubated at 20, 37, and 95 °C for the indicated time points, respectively. The inhibitory activity of NfCPI against cysteine proteases was analyzed. All experiments were performed in triplicate and the mean and standard deviation (SD) values are presented.

Figure 4

Expression pattern of NfCPI in N. fowleri. (A) The morphological changes of N. fowleri during encystation. N. fowleri trophozoites were incubated in encystation medium and morphological changes of the amoeba were analyzed at the indicated time points by microscopy. (B) Semiquantitative RT-PCR. The transcription profiles of nfcpi, nfcb, nfcbl, nfcz, and nfcalpain-like were analyzed during encystation (left panel). Graphs show the mean ± SD densitometric ratios of nfcpi and nfgapdh of three independent experiments (right panel). Significance of the data was analyzed by using one-tailed t test, ** p < 0.005, *** p < 0.001. (C) Immunoblot analysis. Expression profile of NfCPI in trophozoites and cysts was determined by immunoblot with anti-NfCPI MAb or non-immunized mouse IgG. (D) Localization. Immunofluorescence Assay (IFA) was performed with anti-NfCPI MAb. N. fowleri trophozoites and cysts were fixed on cover slip and probed with anti-NfCPI MAb and FITC-conjugated anti-mouse IgG. The slide was observed with a confocal laser scanning microscope.