Proteomic analysis of skin invasion by blood fluke larvae

Abstract

Background: During invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.

Methodology and results: Human skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in "triple play" mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples.

Conclusions: This analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire.

Figure 1. Cercaria and invasive tunnel in epidermis of human skin at 1/2-hour post invasion.

A). The parasite larva is just entering the dermis toward the bottom of the figure. Note tunnel (arrows) formed from destruction of epidermal cells by both acantholysis and apoptosis. It is fluid from these tunnels that was targeted for proteome analysis. B) Model of presumed acquisition of fluid from skin invaded by schistosome cercariae. Proteins from the tunnels produced by cercariae (C), as well as lysed epidermal cells and dermal fluid are identified in Tables 1 and 2.

Figure 2. Tunel assay demonstrating apoptosis of epidermal cells (punctate brown immunoperoxidase staining on left).

A cercaria is immediately above 100-micron bar. It is now in the dermis (D), but had invaded epidermis from left to right. Internal control is absence of apoptotic signal in intact epidermis on right (E).