Calendula officinalis Triterpenoid Saponins Impact the Immune Recognition of Proteins in Parasitic Nematodes

Abstract

The influence of triterpenoid saponins on subcellular morphological changes in the cells of parasitic nematodes remains poorly understood. Our study examines the effect of oleanolic acid glucuronides from marigold (Calendula officinalis) on the possible modification of immunogenic proteins from infective Heligmosomoides polygyrus bakeri larvae (L3). Our findings indicate that the triterpenoid saponins alter the subcellular morphology of the larvae and prevent recognition of nematode-specific proteins by rabbit immune-IgG. TEM ultrastructure and HPLC analysis showed that microtubule and cytoskeleton fibres were fragmented by saponin treatment. MASCOT bioinformatic analysis revealed that in larvae exposed to saponins, the immune epitopes of their proteins altered. Several mitochondrial and cytoskeleton proteins involved in signalling and cellular processes were downregulated or degraded. As possible candidates, the following set of recognised proteins may play a key role in the immunogenicity of larvae: beta-tubulin isotype, alpha-tubulin, myosin, paramyosin isoform-1, actin, disorganized muscle protein-1, ATP-synthase, beta subunit, carboxyl transferase domain protein, glutamate dehydrogenase, enolase (phosphopyruvate hydratase), fructose-bisphosphate aldolase 2, tropomyosin, arginine kinase or putative chaperone protein DnaK, and galactoside-binding lectin. Data are available via ProteomeXchange with identifier PXD024205.

Keywords: TEM nematode ultrastructure; protein patterns; triterpenoid saponins.

Figure 1

The ultrastructure of H. polygyrus bakeri L3 following marigold GlcUAOA treatment. Representative TEM images from morphological observations: Images (A–D)—L3(CTR) as negative controls with no apoptotic changes; Images (E–H)—L3(EtOH) exposed to ethanol; Images (I–N)—L3(GlcUAOA) demonstrating cell, apoptosis, nuclear condensation (white arrows) and cell shrinkage due to apoptosis (black arrows); autophagosome (N); the blue asterix at (G) indicates fragmented myofibrils; Image L—swollen mitochondrion of L3(GlcUAOA). a: alea; bz: basal zone; c: cuticle; cz: cortical zone; ep: epicuticle; h: hypodermis; im: inner membrane; in: intestine; m: mitochondrion; mf: myofibrils; n: nucleus. Scale bar: 2000 nm (A,E,I); 1000 nm (B,C,E–G,J–I) and 500 nm (D,H, L–N).

Figure 2

Morphometric analysis of cell structure in transverse and longitudinal section of H. polygyrus bakeri larvae; the length of myofibrils (A) in longitudinal section, the distance between the cuticle layers (B) in transverse section, the size of mitochondrion (C). Statistics: ANOVA, n = 10, * p < 0.05, ** p < 0.01, *** p < 0.001, mean ± SD.

Figure 3

Elution profile of somatic protein isolated from H. polygyrus bakeri larvae. (A)—negative control L3(CTR); (B)—exposed to 8% ethanol L3(EtOH); (C)—exposed to C. officinalis glucuronides L3(GlcUAOA). A total of 100 µL of antigen solution was separated on a ProteinPak column and eluted isocratically using PBS (pH 7.4) with flow rate 400 µL/min for 45 min.

Figure 4

SDS-PAGE protein pattern stained with Coomassie blue of H. polygyrus bakeri L3 (A) and Western blot protein pattern of L3 (B) recognised by hyperimmune rabbit serum. Proteins extracted from: 1—control larvae L3(CTR), 2—larvae exposed to 8% ethanol L3(EtOH), 3—larvae exposed to C. officinalis glucuronides L3(GlcUAOA): kDa—molecular mass of protein marker. Primary Western blot detection for B is placed in Supplementary Data 1. Red arrows indicate bands analysed by LC-MS/MS.

Figure 5

Functions of H. polygyrus bakeri L3 stage proteins according to Gene Ontology (GO): (A)—proteins categorised by their biological processes; (B)—proteins categorised by their molecular function; (C)—proteins categorised by their component category. Information obtained from UniProtKB and Quick GO databases.