Fig 1.
Amino acid sequences of EgKI-1 (1) and EgKI-2 (2).
A. Signal sequences (18 amino acids) are in red, the Kunitz domain of both proteins is boxed and the Kunitz family signature highlighted in black. The conserved cysteine residues are shown in orange; EgKI-1 has six whereas EgKI-2 has five with one position replaced by a glycine (blue). The P1 reactive sites of both proteins are highlighted in green. B. Schematic diagram of (i) EgKI-1 showing three disulphide bridges, and (ii) EgKI-2 presenting two disulphide bridges.
Fig 2.
A. Partial amino acid sequence comparison of EgKI-1 and EgKI-2 with other Kunitz type protease inhibitors from: Fasciola hepatica (FhKTM, AAB46830.1); BPTI (1510193A); Echinococcus granulosus (EgKU8, ACM79010.1); Ancylostoma ceylanicum (AceKI, AAD51334.1); Conus striatus (Conk-S1, P0C1X2.1); the first domain of Ancylostoma caninum Kunitz inhibitor (Ac-KPI-1, AAN10061.1).
The six conserved cysteine residues are marked by * and the pattern of disulphide bond formation is shown in brackets. The P1 reactive site is marked by the arrow head and the Kunitz family signature by the dashed double head arrow. B. Phylogenetic analysis of EgKI-1 and EgKI-2 with other Kunitz type protease inhibitors: FhKTM, EgKU8, BPTI, SmKI, Conk-S1, human tissue factor pathway inhibitor-2 (TFPI-2, AAA20094), Black fly (Simulium vittatum) (Simukunin, ACH56928.1), Orb weaver spider (Araneus ventricosus) (AvKTI, AFX95921.1), Black mamba (Dendroaspis polylepis) (DendrotoxinK, 1097974).
Fig 3.
Normalized expression levels of the EgKI genes in E. granulosus.
The Y axis represents the number of copies after dividing the number of copies of either gene of interest (GOI) by the number of copies of the house keeping gene (HKG). PSC, protoscoleces; AW, adult worms; HCM, hydatid cyst membrane; ONC, oncospheres. Error bars represent the mean ± SEM.
Fig 4.
(A) SDS-PAGE analysis and Western blotting with mouse antisera raised against (B) EgKI-2 and (C) EgKI-1 proteins: Lane 1, EgKI-1; Lane 2, EgKI-2; Lane 3, BPTI; Lane 4, UTI; Lane 5, SmKI; Lane M, protein marker. Aliquots of 3 μg from each protein sample were used for SDS-PAGE and 0.5 μg for western blotting.
Fig 5.
Immunolocalization of EgKI-2 in histological sections of an adult worm of E. granulosus (A) Bright field, (B) probed with pre-immune sera as negative control, (C) probed with EgKI-2 immunized mouse serum (1:200).
DAPI counterstained nuclei are stained blue and the positive green fluorescence marks the presence of EgKI-2 along the tegument (t). Scale Bar = 100 μm.
Fig 6.
(A) Predicted calcium binding site of EgKI-1, shown in blue. (B) Calcium binding assay: SDS-PAGE gel (left) and corresponding western blot membrane of the calcium binding assay showing clear bands corresponding to the EgKI-1 protein: M-Marker; 1, 6 μg EgKI-1; 2, 2 μg EgKI-1; 3, 6 μg EgKI-2; 4, 6 μg BSA.
Table 1.
Inhibitor constant (Ki) values in Molar (M) range of EgKI-1 and EgKI-2.
Fig 7.
Inhibition of different serine proteases with increasing concentrations of the EgKI proteins.
The relative activity (as a %) with: (A) nanomolar (nM) concentration range of EgKI-1 with trypsin, pancreatic elastase and cathepsin G, and EgKI-2 with trypsin (B) picomolar (pM) concentration range of EgKI-1 with neutrophil elastase and chymotrypsin. (C) Progress curves for trypsin inhibition with increasing concentrations of EgKI-2 (0 nM, 10 nM, 20 nM, 100 nM) with different substrate concentrations ([S]).
Fig 8.
Inhibition of neutrophil chemotaxis by EgKI-1 in the mouse air pouch model.
The data represent the mean ± SEM values of groups of eight mice in two independent experiments and analysis by one-way ANOVA. * P value, 0.03; ** P value, 0.001; ns, not significant (p value ≥ 0.05).