Proteomic analysis of human skin treated with larval schistosome peptidases reveals distinct invasion strategies among species of blood flukes

Abstract

Background: Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts.

Methods: Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays.

Conclusions: This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts.

Figure 1. Differential expansion of select peptidase gene families in schistosome species.

Phylogenetic analysis of cercarial elastase (A) and cathepsin B2 (B) protein sequences reveals expansion of each gene family in different lineages of schistosomes. Boxes indicate proteins previously determined to be present in cercarial secretions, as determined by LC MS/MS.

Figure 2. Human collagen I is preferentially cleaved by SmCB2.

In vitro cleavage of human collagen I confirms ex vivo analysis, and reveals differential cleavage by CE and CB2. (A) SmCB2 digestion of collagen I (B) SmCE digestion of collagen I. Digestion reactions were performed for 1–22 hours at 37°C. * indicates pre-incubation with (A) 1 mM CAO74 or (B) 1 mM AAPF-CMK. ** indicates no peptidase control. Arrows indicate bands submitted for Edman degradation. (C) Schematic of human collagen I, indicating cleavage sites as determined by Edman degradation (VWC-Von Willenbrand Factor Type C domain; COL- collagen triple helix repeat).

Figure 3. Human complement C3 is cleaved by both SmCE and SmCB2.

In vitro cleavage of human complement C3 protein confirms ex vivo analysis, and reveals differential cleavage by CE and CB2. (A) SmCB2 digestion of complement C3. (B) SmCE digestion of complement C3. Digestion reactions were performed for 1–22 hours (SmCB2) or 1–5 hours (SmCE) at 37°C. * indicates pre-incubation with (A) 1 mM CAO74 or (B) 1 mM AAPF-CMK. ** indicates no peptidase control. Arrows indicate bands submitted for Edman degradation. (C) Schematic of human complement C3, indicating cleavage sites as determined by Edman degradation (MGI-macroglobulin-1; A2M-alpha-2-macroglobulin; ANA-.Anaphylatoxin homologous domain; NTR-netrin-like domain).