Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis

Abstract

Background: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs).

Methods: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography.

Results: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba.

Conclusions: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.

Keywords: Acanthamoeba; Host‐pathogen interaction; Human corneal epithelial cells; Mannose-binding protein; Metalloproteinase; Pathogenesis; Protease inhibitor; Serine protease; Virulence factors.

Fig. 1

Main classes and general structures of protease inhibitors used (source structures ChemSpider)

Fig. 2

Profiles of extracellular proteases produced by Acanthamoeba and HCECs in monoculture and after 1 h of co-culture. The molecular masses are indicated on the left side of each gelatin zymography (10% polyacrylamide). A and B Proteolytic profiles of Acanthamoeba 1BU and SIN20 strains without and during contact with HCECs. C Proteolytic profiles of the newly generated subset of strains 1BUH3x and SIN20H3x, without and during contact with HCECs (the red arrow represents the new band displayed by SIN20H3x after reactivation of the attenuated properties, the white arrows the additional proteases displayed after contact with HCECs, and blue arrows proteases released by HCECs)

Fig. 3

In vitro secretion kinetics of proteases secreted by 1BUH3x (A) and SIN20H3x (B) after contact with HCECs using 1D in-gel zymography. The patterns of extracellular proteases displayed by HCECs and Acanthamoeba in monoculture were used as experimental controls

Fig. 4

Electrophoretic profiles of Acanthamoeba strains SIN20H3x (A) and 1BUH3x (B) and dose-dependent inhibitory effects of PMSF, E-64, PHE and TLCK. A Arrows indicate the absence of two bands after exposure to phenanthroline (PHE)

Fig. 5

Specificities of proteases secreted by Acanthamoeba during contact with HCECs. Proteolytic patterns of Acanthamoeba strains SIN20H3x (A) and 1BUH3x (B) after 1 h of co-culture with HCECs and HCEC lysate

Fig. 6

Effect of the protein synthesis inhibitor cycloheximide (100 µM) on the secretion of extracellular proteases produced by HCECs and 1BUH3x in mono- and co-culture. The arrow shows the prominence of a protease band produced by HCECs after 1 h of exposure to cycloheximide

Fig. 7

Expression of the MBP genes in Acanthamoeba 1BUH3x before (black bar) and after (grey bars) contact with HCECs. The y-axis indicates the fold increase in mRNA levels compared to the control (without contact). The error bars show the standard deviation of the mean (SEM). Values were obtained from at least three biological replicates in duplicate. ns: non-significant according to statistical analysis (Student's t-test)

Fig. 8

Viability of untreated and PMSF-treated (250 µM) HCECs co-cultured with Acanthamoeba 1BUH3x and SIN20H3x. The time of exposure to the amoeba or/and the inhibitor was 8 h. Values represent the mean of four independent experiments, each in triplicate. Statistical analysis was performed through two-way ANOVA with Dunnett’s multiple comparisons test (*P < 0.01, **P < 0.001, ***P < 0.0001 and ns: non-significant)

Fig. 9

Phase contrast microscopy of Acanthamoeba 1BUH3x in monoculture and co-culture with HCECs in the presence or absence of PMSF. A HCECs in monoculture; B 1BUH3x attached to the flask and bound to HCECs; C Acanthamoeba (aggregate, upper part) in co-culture with HCECs, forming clusters and not attaching and binding on the flask and the mammalian cells because of the presence of the inhibitor; D aggregation of amoebae in monoculture due to the presence of PMSF. Scale bar: 50 µm