Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles

Abstract

Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates.

Keywords: Excretion; Fluke; Inhibitor; Parasite; Protease; Secretion.

Figure 1. Detection of proteolytic activity in the ES products of S. mansoni

A. ES products from schistosomula (blue), adult worms (red) and eggs (green) were incubated for 15 to 1,200 min with a 14-mer peptide library. LC-MS/MS sequencing was used to detect the appearance of peptidase cleavage sites B. A sample 14-mer peptide illustrating the complexity of peptidase cleavage. The position and time (minutes) that cleavage products were first detected are indicated. Amino acids are shown in single letter code. Lowercase “n” corresponds to norleucine (Nle). C. Venn Diagram showing the number of unique and shared cleavage sites. D. Spatial distribution of cleavage sites within the 14-mer peptide scaffold.

Figure 2. Generation of a proteolytic specificity signature in the ES products of S. mansoni at different life cycle stages

Generation of a substrate specificity iceLogo signature for A. schistosomule, B. adult and C. egg secretions following 240 or 1,200 minutes incubation with the 14-mer peptide library. Amino acids colored black are significantly increased (p<0.05) in the position relative to a control dataset that corresponds to all possible cleavage sites. Amino acids above the X-axis are found at the given position with higher frequency than the control dataset while amino acids below the axis are rarely or never found at the given position. IceLogo signature showing the substrate specificity profiles of conditioned water from D. uninfected and E. S. mansoni infected Biomphalaria glabrata.

Figure 3. Quantitation of proteolytic activity in ES products of S. mansoni at different life cycle stages

Proteolytic activity in the conditioned media was detected using internally quenched fluorescent substrates. Amino acids are described in single letter code and lowercase ‘t’ corresponds to tert-butyl glycine.

Figure 4. Use of class-specific inhibitors to characterize peptidase activity and specificity in schistosomula secretion

A. ES products were assayed with the internally quenched tQASSRS substrate in the presence of class-specific inhibitors. B. Use of a sample 14-mer peptide to illustrate the time dependent changes in cleavage site accumulation in the presence of class-specific inhibitors compared to a non-inhibited DMSO control. C. An iceLogo substrate profile generated from all cleavage sites in the 14-mer peptide library that were sensitive to AEBSF.

Figure 5. Use of class-specific inhibitors to characterize peptidase activity and specificity in adult secretion

A. ES products were assayed with the internally quenched tQASSRS substrate in the presence of class-specific inhibitors. B. An iceLogo substrate profile consisting of all cleavage sites in 14-mer peptides that were sensitive to AEBSF treatment. C. An iceLogo substrate profile consisting of all cleavage sites in 14-mer peptides that were sensitive to 1,10-Phenanthroline treatment. D. Analysis of the di-aminopeptidase specificity detected in adult ES products. The bar graph represents the time interval at which each the cleavage product was first detected.

Figure 6. Use of class-specific inhibitors to characterize peptidase activity and specificity in egg secretion

A. ES products were assayed with the internally quenched tQASSRS substrate in the presence of class-specific inhibitors. B. An iceLogo substrate profile consisting of all cleavage sites in the 14-mer peptides that were sensitive to AEBSF treatment. C. An iceLogo substrate profile consisting of all cleavage sites in the 14-mer peptides that were sensitive to E-64 treatment. D. A Venn diagram showing that many of the cleavage sites are resistant to both AEBSF and E-64. E. ES products were assayed with the internally quenched GRFGVWKA substrate in the presence of class-specific inhibitors. F. An iceLogo substrate profile consisting of all cleavage sites in the 14-mer peptides that were sensitive to 1,10-Phenanthroline treatment.