BACKGROUND: Blood flukes (Schistosoma spp.) are parasites that can survive for years or decades in the vasculature of permissive mammalian hosts, including humans. Proteolytic enzymes (proteases) are crucial for successful parasitism, including aspects of invasion, maturation and reproduction. Most attention has focused on the 'cercarial elastase' serine proteases that facilitate skin invasion by infective schistosome larvae, and the cysteine and aspartic proteases that worms use to digest the blood meal. Apart from the cercarial elastases, information regarding other S. mansoni serine proteases (SmSPs) is limited. To address this, we investigated SmSPs using genomic, transcriptomic, phylogenetic and functional proteomic approaches. METHODOLOGY/PRINCIPAL FINDINGS: Genes encoding five distinct SmSPs, termed SmSP1 - SmSP5, some of which comprise disparate protein domains, were retrieved from the S. mansoni genome database and annotated. Reverse transcription quantitative PCR (RT- qPCR) in various schistosome developmental stages indicated complex expression patterns for SmSPs, including their constituent protein domains. SmSP2 stood apart as being massively expressed in schistosomula and adult stages. Phylogenetic analysis segregated SmSPs into diverse clusters of family S1 proteases. SmSP1 to SmSP4 are trypsin-like proteases, whereas SmSP5 is chymotrypsin-like. In agreement, trypsin-like activities were shown to predominate in eggs, schistosomula and adults using peptidyl fluorogenic substrates. SmSP5 is particularly novel in the phylogenetics of family S1 schistosome proteases, as it is part of a cluster of sequences that fill a gap between the highly divergent cercarial elastases and other family S1 proteases. CONCLUSIONS/SIGNIFICANCE: Our series of post-genomics analyses clarifies the complexity of schistosome family S1 serine proteases and highlights their interrelationships, including the cercarial elastases and, not least, the identification of a 'missing-link' protease cluster, represented by SmSP5. A framework is now in place to guide the characterization of individual proteases, their stage-specific expression and their contributions to parasitism, in particular, their possible modulation of host physiology.

Center for Discovery and Innovation in Parasitic Diseases Department of Pathology University of California San Francisco San Francisco California United States of America

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic; Department of Parasitology Faculty of Science Charles University Prague Prague Czech Republic

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic; Institute of Parasitology Biology Centre Academy of Sciences of the Czech Republic Ceske Budejovice Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague Czech Republic

Institute of Parasitology Biology Centre Academy of Sciences of the Czech Republic Ceske Budejovice Czech Republic

Institute of Parasitology Biology Centre Academy of Sciences of the Czech Republic Ceske Budejovice Czech Republic; Fraunhofer Institute for Molecular Biology and Applied Ecology IME Project Group Bioresources Gießen Germany

Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Hinxton United Kingdom

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GENBANK
CAA09691, KF535923

PDB
1JRT