Nf-GH, a glycosidase secreted by Naegleria fowleri, causes mucin degradation: an in vitro and in vivo study

Abstract

Aim: The aim of this work was to identify, characterize and evaluate the pathogenic role of mucinolytic activity released by Naegleria fowleri.

Materials & methods: Zymograms, protease inhibitors, anion exchange chromatography, MALDI-TOF-MS, enzymatic assays, Western blot, and confocal microscopy were used to identify and characterize a secreted mucinase; inhibition assays using antibodies, dot-blots and mouse survival tests were used to evaluate the mucinase as a virulence factor.

Results: A 94-kDa protein with mucinolytic activity was inducible and abolished by p-hydroxymercuribenzoate. MALDI-TOF-MS identified a glycoside hydrolase. Specific antibodies against N. fowleri-glycoside hydrolase inhibit cellular damage and MUC5AC degradation, and delay mouse mortality.

Conclusion: Our findings suggest that secretory products from N. fowleri play an important role in mucus degradation during the invasion process.

Keywords: Naegleria fowleri; glycosidases; mucinases; mucins; secretory products.

Figure 1.. Proteolytic and mucinolytic patterns evaluated at various pH values.

(A) Gelatin–zymogram showing proteolytic bands from secretory products at pH 5, 7, and 9; lanes 1–3, respectively. (B) Bovine submaxillary mucin–zymogram showing mucinolytic activities of ethanol-treated secretory products at pH 5 (lane 1: >250 and 94 kDa) and pH 7 (lane 2: >250, 94 and 53 kDa). Mucinolytic activities were not evident at pH 9 (lane 3). Bactocasitone medium at pH 7 (100 mM Trizma and 2 mM CaCl₂) was used as a negative control (lane 4). All activities were evaluated at 37°C.

Figure 2.. Cysteine and serine protease inhibitors decrease mucinolytic activities.

(A) Ethanol-treated secretory products were incubated with protease inhibitors and evaluated in bovine submaxillary mucin–zymograms as follows: ethanol-treated secretory products without inhibitor (lane 1), or with 10 mM pHMB (lane 2), 10 μM E-64 (lane 3), 5 mM NEM (lane 4), 5 μM IAA (lane 5), 5 mM PMSF (lane 6), aprotinin (lane 7), or 10 mM 1,10-phenanthroline (lane 8). All the zymograms were activated at pH 7, the results correspond a one gel with several lanes. (B) Densitometric analyses of >250-kDa (white bars), 94-kDa (black bars) and 53-kDa (gray bars) protein activities using ImageJ software, graphed by GraphPad Prism and expressed as ROD values. Statistical analysis was performed by two-way ANOVA comparing the degradation without inhibitors. Bars display the mean ± SE of three independent assays. *p < 0.05. E-64: 1-{N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino}-4-guanidinobutane); IAA: Iodoacetamide; NEM: N-ethylmaleimide; pHMB: p-hydroxymercuribenzoate; PMSF: Phenyl-methyl sulfonyl fluoride; ROD: Relative optical density.

Figure 3.. Secretion of an inducible Naegleria fowleri mucinase of 94 kDa using BSM.

(A) N. fowleri trophozoites were exposed to 0.1 mg/ml BSM for 1, 3, 6 and 12 h, and the ethanol-treated secretory products from N. fowleri were tested in a BSM–zymogram. Activity at 94 kDa was detected at all times evaluated (lanes 1–4). SPs from nonstimulated trophozoites revealed bands of >250, 94 and 53 kDa, but only at 6 and 12 h (lanes 7 & 8). (B) Statistical analysis was performed by two-way ANOVA comparing degradation without inhibitors. All bars display the mean ± SE of three independent assays. *p < 0.05. BSM: Bovine submaxillary mucin; ROD: Relative optical density.

Figure 4.. Degradation of glycosidic substrates by glycosidases from Naegleria fowleri.

(A) SDS-PAGE 10%: molecular weight marker (lane 1), ethanol-treated SPs (lane 2) and MEF (lane 3) samples. BSM–zymogram of ethanol-treated SPs (lane 3) and MEF (lane 4) samples. (B) Activity of ethanol-treated SPs (solid squares) and MEF (solid triangles) on β-galactopyranoside. (C) Activity of ethanol-treated SPs (solid squares) and MEF (solid triangles) on β-glucopyranoside. (D) Activity of ethanol-treated SPs (solid squares) and MEF (solid triangles) on β-mannopyranoside. For each substrate, ethanol-treated SPs and MEF were also pretreated with 10 mM pHMB (empty squares and empty triangles, respectively). Enzymatic activities were analyzed at various pH values and determined spectrophotometrically at 405 nm. Statistical analysis two-way ANOVA was performed, and the data are reported as the means ± SEM of three independent experiments. *p < 0.05. MEF: Mucinase-enriched fraction; pHMB: p-hydroxymercuribenzoate; SP: Secretory product.

Figure 5.. Identification of Nf-GH in Naegleria fowleri trophozoites using antibodies against glycoside hydrolase.

(A) 3D prediction of GH showing the selected peptide sequence (blue) to obtain the specific GH antibodies (anti-GH). (B) Detection of Nf-GH in ethanol-treated secretory products and mucinase-enriched fraction. Wb assays: secretory products (lane 1) and mucinase-enriched fraction (lane 2) samples; a specific band at 94 kDa was detected by the anti-GH antibodies. The rabbit PI serum was tested in secretory products and mucinase-enriched fraction (lanes 3 & 4) as experimental controls (C) Localization of Nf-GH in N. fowleri trophozoites under nonpermeabilized conditions. (D) Localization of Nf-GH in N. fowleri trophozoites under permeabilized conditions; the amoebae were permeabilized with 0.2% Triton X-100. Anti-GH recognition was detected using a secondary FITC-conjugated antibody, whereas nuclei were labeled with propidium iodide. The PI serum was used as a control. Images were obtained by confocal microscopy (Carl Zeiss, LMS 700) at 60×, scale bar, 10 μm. GH: Glycoside hydrolase; Nf-GH: GH of Naegleria fowleri; PI: Preimmune.

Figure 6.. Protective effect of anti-GH antibodies on NCI-H292 cell death induced by Naegleria fowleri trophozoites.

(A) N. fowleri trophozoites co-incubated with NCI-H292 during 1, 3 and 6 h in the absence or presence of anti-GH antibodies (1.5 μg/ml). Anti-GH antibodies prevent cell death even at 6 h. NCI-H292 cells without trophozoites were used as a negative control, while cells treated with 0.2% Triton X-100 were used as a positive control. Cell viability was evaluated using SYTOX green, which marks dead cells in green. Images were obtained with a fluorescent inverted Nikon microscope (eclipse Ti-U) at 20×. (B) Quantification of the fluorescence intensity of labeled nuclei using ImageJ software. All bars display the mean ± SE of three independent experiments. *p < 0.001. GH: Glycoside hydrolase; Nf: Naegleria fowleri.

Figure 7.. Naegleria fowleri GH causes MUC5AC degradation.

(A) Detection of MUC5AC in secreted mucin from NCI-H292 cells. SDS-PAGE: molecular weight marker (lane 1), secreted mucin from NCI-H292 cells (lane 2), two evident bands at >250 kDa and 40 kDa are shown. Western blot: Detection of the >250-kDa protein of secreted mucin from NCI-H292 cells using the anti-MUC5AC antibody (lane 3). (B) Zymogram copolymerized with secreted mucins from NCI-H292 cells was used as substrate. MEF loaded alone (lane 1), MEF preincubated with anti-GH antibodies (lane 2) or MEF preincubated with pHMB inhibitor (lane 3); gels were activated at pH 7 and 37°C. Mucinolytic activity at approximately 94 kDa by Naegleria fowleri GH was observed (lane 1). (C) Densitometric analysis of panel A was performed using ImageJ and GraphPad Prism software; *p < 0.05. (D) Detection of MUC5AC degradation by MEF by dot-blot (dot 1), secreted mucins from mucoepithelial cells without treatment; (dot 2) secreted mucins treated with PK, (dot 3) secreted mucins treated with 94-kDa fraction, (dot 4) secreted mucins treated with Naegleria fowleri GH preincubated with pHMB, (dot 5) secreted mucins treated with MEF preincubated with anti-GH antibodies, (dot 6) secretory products without secreted mucins, used as a negative control. All dot-blots were developed using antibodies against the MUC5AC mucin. The load protein was stained with Ponceu's solution. (E) Densitometric analysis of MUC5AC degradation of the assays shown in panel D was performed using ImageJ software. Bars display the mean ± SE of three independent assays. *p < 0.05. GH: Glycoside hydrolase; MEF: Mucinase-enriched fraction; pHMB: p-hydroxymercuribenzoate; PK: Proteinase K; ROD: Relative optical density.

Figure 8.. Mouse mortality by primary amoebic meningoencephalitis was delayed using anti-GH antibodies.

Mouse mortality was determined in animals instilled with amoebae (12 × 10⁴; triangles) and amoebae preincubated with anti-GH antibodies (30 μg/ml) (squares). Noninfected animals were used as negative controls (diamonds). Infected mice with trophozoites showed mortality 7 days postinfection, while mouse mortality was delayed using anti-GH antibodies for 17 days. The noninfected group survived the entire experiment. Survival curves were analyzed by the log rank test using GraphPad Prism (n = 6, per group). *p < 0.001. GH: Glycoside hydrolase.