Research Article: Proteomic Analysis of Skin Invasion by Blood Fluke Larvae

Date Published: July 16, 2008

Publisher: Public Library of Science

Author(s): Elizabeth Hansell, Simon Braschi, Katalin F. Medzihradszky, Mohammed Sajid, Moumita Debnath, Jessica Ingram, K. C. Lim, James H. McKerrow, Malcolm K. Jones

Abstract: BackgroundDuring 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 ResultsHuman 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.ConclusionsThis 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.

Partial Text: Human skin is a highly evolved barrier to environmental pathogens [1]. Flattened, keratinized epidermal cells, made adherent by a lipid-rich intercellular milieu, constitute the most superficial barrier called the stratum corneum. This cornified layer of cells is derived from keratinized cells in the underlying stratum granulosum. Beneath this layer, large glycogen-rich epidermal cells form a cellular matrix called the stratum spinosum. Cells in the stratum spinosum are locked together by multiple dense intercellular bridges formed by protein complexes called desmosomes. The epidermis is also anchored to a basement membrane that separates it from the dermis [1].

This study identified proteins in human skin that are relevant to the biology of the host-parasite interplay occurring during initial infection of the human host by S. mansoni cercariae. The results validate, clarify, or extend analyses of invasive behavior of cercariae observed in vitro, and present a molecular view of invasion of skin by a human parasite in as “biological” a situation as is ethically possible. That said there are likely other relevant protein factors that might not be present due to ethical limitations. First, while this is recently harvested human skin, it is no longer perfused from the vascular system. Components of the bloodstream that might reach the site of invasion by chemotaxis may not be present. The resolution of mass spectrometry following tryptic digest may also not identify peptide fragments generated from host proteins that are targets for cercarial protease, or other protease activity. Nevertheless, the rich dataset of both parasite and host skin proteins, discussed below, provides the most complete picture to date of the pathobiology of invasion. The experiment was performed twice (only one experiment is reported here), and control skin samples incubated in water in an identical manner ensured that host proteins identified as bound or fragmented by larval invasion were not present due to nonspecific skin degeneration (Table 2). Indeed the abundant proteins induced in UV-stressed or heat-stressed skin in previous studies [29] were either not abundant (HSP 70) or even present (HSP 27) in these skin samples. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled (Figure 1A,B).

Source:

http://doi.org/10.1371/journal.pntd.0000262

 

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