Fasciola hepatica Cathepsin L Zymogens: Immuno-Proteomic Evidence for Highly Immunogenic Zymogen-Specific Conformational Epitopes to Support Diagnostics Development

Abstract

Fasciola hepatica, the common liver fluke and causative agent of zoonotic fasciolosis, impacts on food security with global economic losses of over $3.2 BN per annum through deterioration of animal health, productivity losses, and livestock death and is also re-emerging as a foodborne human disease. Cathepsin proteases present a major vaccine and diagnostic target of the F. hepatica excretory/secretory (ES) proteome, but utilization in diagnostics of the highly antigenic zymogen stage of these proteins is surprisingly yet to be fully exploited. Following an immuno-proteomic investigation of recombinant and native procathepsins ((r)FhpCL1), including mass spectrometric analyses (DOI: 10.6019/PXD030293), and using counterpart polyclonal antibodies to a recombinant mutant procathepsin L (anti-rFhΔpCL1), we have confirmed recombinant and native cathepsin L zymogens contain conserved, highly antigenic epitopes that are conformationally dependent. Furthermore, using diagnostic platforms, including pilot serum and fecal antigen capture enzyme-linked immunosorbent assay (ELISA) tests, the diagnostic capacities of cathepsin L zymogens were assessed and validated, offering promising efficacy as markers of infection and for monitoring treatment efficacy.

Keywords: cathepsin; diagnostics; fasciolosis; recombinant; triclabendazole.

Figure 1

1-DE of recombinant mutant F. hepatica procathepsin L1 (rFhΔpCL1) and immunoreactivity against polyclonal anti-rFhΔpCL1 IgG. (A) 2 μg rFhΔpCL1 was analyzed by 1-DE, and the intact zymogen fragment (boxed) was excised and analyzed by LC-MS² (Table 1). Two hits were consistent between duplicate sample submissions (procathepsin L1 chain A, 2O6X_A; cathepsin L-like proteinase, ADP09371.1), including peptide recovery from pro-peptide (16–105 aa) and cathepsin L protease (106–326 aa/TERM) regions. (B) 1 μg rFhΔpCL1 was probed with 1:800–1:30,000 pre- (*) and post-immunization (**) rabbit sera and detected by alkaline phosphatase-conjugated anti-rabbit IgG raised in goat. Abbreviations: MW, Amersham Low Molecular Weight SDS Calibration Kit (Mr); FhΔpCL1², dimer-sized protein; and FhpCL1-SP, procathepsin with cleaved signal peptide.

Figure 2

1-DE of recombinant wild-type F. hepatica procathepsin L1 (rFhpCL1^WT) and immunoreactivity against polyclonal anti-rFhΔpCL1 IgG. (A) 5 μg of rFhpCL1^WT was analyzed by 1-DE, including inactivated protein and following autocatalysis into cathepsin L protease and cleaved pro-enzyme peptides. Two rFhpCL1^WT zymogen fragments of approximately 35 and 37 kDa (boxed) were excised and analyzed by LC-MS² (Table 1), confirming one hit consistent between duplicate analysis (secreted cathepsin L1, AAB41670.2). (B) 0.5 μg rFhpCL1^WT from each autocatalysis time point was probed with 1:15,000 anti-rFhΔpCL1 polyclonal rabbit sera alongside 0.5 μg rFhΔpCL1(+) and detected by alkaline phosphatase-conjugated anti-rabbit IgG raised in goat. Abbreviations: MW, Amersham Low Molecular Weight SDS Calibration Kit (Mr); (p)pCL, (pre-)procathepsin L; I, intermediates proteins; and CL, cathepsin L protease.

Figure 3

1-DE of recombinant F. hepatica procathepsin L1 (rFhpCL1) and immunoreactivity against polyclonal anti-rFhΔpCL1 IgG. (A) 20 μg rFhpCL1 was analyzed by 1-DE, and six protein fragments (boxed: 1–6) between ≈14 to 37 kDa were excised and analyzed by LC-MS² (Table 1). One hit was consistent in all fragments for cathepsin protein CatL1-MM3p partial (CCA61803.1), and a further hit was found for fragments 3 and 6 (cathepsin L-like proteinase, ADP09371.1) and fragment 6 (cathepsin L, BAB86959.1) only. Peptide recovery between CCA61803.1 and ADP09371.1 hits included pro-peptide (16–105) and cathepsin L protease (106–326) regions, whereas BAB86959.1 peptides pertained to the protease (106–324) region only. (B) 2 μg rFhpCL1 was probed with 1:10,000 anti-rFhΔpCL1 polyclonal rabbit sera alongside 0.05 μg rFhΔpCL1(+) and detected by alkaline phosphatase-conjugated anti-rabbit IgG raised in goat. Abbreviations: MW, Amersham Low Molecular Weight SDS Calibration Kit (Mr); x-mers, dimer- and trimer-sized proteins.

Figure 4

Representative 2-DE of in vitro-cultured live and dead adult F. hepatica excretory/secretory (ES) CL zymogen sub-proteomes and immunoreactivity against polyclonal anti-rFhΔpCL1 IgG. 25 μg ES products of live untreated (Ai) and dead (ethyl 4-aminobenzoate-terminated) (Bi) adult F. hepatica were analyzed by 2-DE. The area consisting of cathepsin L zymogens (≈30 to 38 kDa and 5.2–7.8 pI, boxed) were excised and analyzed by LC-MS² (Table 2). 25 μg 2-DE-separated ES products of live untreated (Aii) and dead (ethyl 4-aminobenzoate-terminated) (Bii) adult F. hepatica were probed with anti-rFhΔpCL1 diluted to 1:5000. The greatest antigenicity was observed in protein spots separating at the same position as procathepsin L (pCL) and minor immunoreactivity of proteases (CL) in these native samples. Abbreviations: MW, Amersham Low Molecular Weight SDS Calibration Kit (M_r).

Figure 5

Validation of F. hepatica procathepsin L-based ELISA platforms for the comparison of antigen immunogenicity and capture during infection with TCBZ-S or TCBZ-R F. hepatica strains. Adjusted average ODs were calculated from two duplicate ELISA tests for both serum or fecal antigen capture ELISA platforms. (A) FhΔpCL1 Ag-ELISA was validated for serum antibody detection, whereby rFhΔpCL1 [0.5 μg/mL] was detected by experimental infection sera (1:750, n = 3 sheep, one parasite strain each) from 0–17 weeks post infection (wpi) with TCBZ-S (Aberystwyth, Italian, Miskin: dashed line) or TCBZ-R (Kilmarnock, Penrith, Stornoway: solid line) F. hepatica and following clinical administration of TCBZ at 12 wpi. Positive OD values for each sera group were considered when exceeding the cutoff (C–O), shown as one standard deviation above the negative Ag (BSA) OD score (dot line, TCBZ-S: 0.0901; dot-dash line, TCBZ-R: 0.0815). (B) Anti-rFhΔpCL1 IgG sandwich ELISA was validated for F. hepatica fecal antigen capture and identification of treatment success using anti-rFhΔpCL1 polyclonal IgG for capture and detection. Sheep fecal samples pooled from experimental infection fecal samples (n = 2 sheep, one parasite strain each) from 0–17 wpi, including TCBZ-S: Aberystwyth or Italian strains, or TCBZ-R: Kilmarnock or Stornoway strains. Positive OD values were considered when exceeding the −0.04329 OD cutoff (C–O; dot line), shown as one standard deviation above the highest average OD value measured for uninfected sheep samples. Error bars are one standard deviation above and below average ODs, and the shaded line indicates the time point of TCBZ administration.

Figure 6

Dot blot analysis of IgG immunoreactivity of helminth-infected livestock serum against rFhΔpCL1. rFhΔpCL1 (0.01 μg/dot) was probed with pooled whole serum diluted to 1:700 (n = 2 sheep, with either C. daubneyi, H. contortus, or O. circumcinta), 1:100 (n = 2 cattle, with C. oncophora infection), or 1:5000 (n = 2 rabbits immunized with anti-rFhΔpCL1), and IgG binding was detected using anti-sheep, anti-cattle, or anti-rabbit IgG at 1:30,000 per appropriate sample and the BCIP-NBT system until a precipitant appearance in the positive control. Negative controls include: 1^–, pre-[rFhΔpCL1] immunization; 2^–, anti-bovine (2° antibody only); 3^–, anti-sheep (2° antibody only); and 4^–, anti-rabbit (2° antibody only). The asterisk (*) indicates these sera were collected at day 39 (between 5–6 wpi). Abbreviations: +, positive control; −, negative control; and wpi, week(s) post infection.