Comparative proteome analysis of the tegument of male and female adult Schistosoma mansoni

Abstract

The tegument, as the surface layer of adult male and female Schistosoma spp. represents the protective barrier of the worms to the hostile environment of the host bloodstream. Here we present the first comparative analysis of sex-specific tegument proteins of paired or virgin Schistosoma mansoni. We applied a new and highly sensitive workflow, allowing detection of even low abundance proteins. Therefore, a streptavidin-biotin affinity purification technique in combination with single pot solid-phase enhanced sample preparation was established for subsequent LC-MS/MS analysis. We were able to identify 1519 tegument proteins for male and female virgin and paired worms and categorized them by sex. Bioinformatic analysis revealed an involvement of female-specific tegument proteins in signaling pathways of cellular processes and antioxidant mechanisms. Male-specific proteins were found to be enriched in processes linked to phosphorylation and signal transduction. This suggests a task sharing between the sexes that might be necessary for survival in the host. Our datasets provide a basis for further studies to understand and ultimately decipher the strategies of the two worm sexes to evade the immune system.

Figure 1

Specifically bound biotin tags the outer surface of adult Schistosoma mansoni. Images obtained by fluorescence microscopy showing representative surface biotinylation of male_single, male_pair, female_single and female_pair adult worms. The worms were incubated with biotin and cross cryosections were then prepared and probed with avidin-FITC (green). Nuclei were stained with DAPI (blue). The specific avidin-FITC signal was detected only in the biotinylated surface tegument in all groups. The difference in size of biotinylated male_single (compared to other male groups) is only due to a different position at which the cryosection was performed.

Figure 2

Research workflow showing the identification process of proteins of adult Schistosoma mansoni. Surfaces of paired and virgin adult schistosomes were biotinylated to extract surface proteins at host-parasite interface. Tegument samples were processed with the Pierce Cell Surface Protein Isolation Kit to isolate biotinylated proteins. Proteins of whole and stripped worms were obtained by their homogenization. Protein extracts were prepared for following LC–MS/MS via single pot solid-phase enhanced sample preparation protocol and protein candidates were identified over database search followed by differential analysis. Thus, gender-specific proteins of adult worms are presented in this study regardless of their stage of development.

Figure 3

Identified proteins in adult Schistosoma mansoni proteomes. Venn diagrams representing the total amount (a) and the group-specific amount (b) of biotinylated tegument proteins (green), non-biotinylated tegument proteins (grey) and proteins of stripped worms (orange) in different adult Schistosoma mansoni worm proteomes.

Figure 4

Identified male- and female-specific proteins of adult Schistosoma mansoni. The numbers and intersections of the identified proteins from different Schistosoma mansoni proteomes was visualized using an Upset plot. Only proteins from S. mansoni were included. Connected dots display shared proteins between datasets, and the total number of proteins identified in a particular dataset is indicated in the set size. Number of male- and female-specific proteins after subtraction of the control groups non-biotinylated tegument proteins (= neg. control) and stripped worm proteins, shown in red.