The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti

Abstract

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S2 subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

Avian Schistosomes and Outbreaks of Cercarial Dermatitis

“…On the other hand, a papain-like cysteine peptidase, termed cathepsin B2, was found in the postacetabular penetration glands of T. regenti. This enzyme was shown to cleave proteins of the host skin, similar to the case with S. japonicum (187,188). Its expression was even higher in intravertebrate stages (schistosomula and adults), suggesting that there are multiple roles of this enzyme during the life cycle (189).…”

Cathepsins B1 and B2 in the neuropathogenic schistosome Trichobilharzia regenti: distinct gene expression profiles and presumptive roles throughout the life cycle

“…All this probably requires understandably more diverse repertoire of transcripts and/or higher transcription rates. The abundancy of TrCB1.1 transcripts correlates with the widely accepted assumption that cathepsin B1 is one of the main digestive enzymes of schistosomula and adults of schistosomes (Dvořák et al 2005;Caffrey et al 2004).…”

“…In our previous work, we have suggested that TrCB2 could be involved in the cercarial invasion of the final host skin (Dolečková et al 2009). The 3.6-fold higer transcription of TrCB2 gene (Table 1) in intramolluscan stages is in accord with this hypothesis.…”

“…Cathepsin B2 (TrCB2) was found as a transcript in the complementary DNA (cDNA) library of sporocysts with developing cercariae and by means of Western blotting in protein extracts of cercariae and schistosomula. Immunohistochemistry showed the presence of TrCB2 in the post-acetabular penetration glands of free-living cercariae (Dolečková et al 2007(Dolečková et al , 2009). Functions of TrCB2 remain unclear; however, since it expressed a higher hydrolytic activity toward skin proteins than hemoglobin and because cercariae possess only a rudimental caecum and do not feed during their short life in aquatic environment, TrCB2 was suggested to be involved in host skin invasion rather than nutrient digestion.…”

“…In the case of cathepsin B1 (TrCB1), six isoforms were identified and the enzyme was localized to the gut of schistosomula, implying its function in nutrient digestion (Dvořák et al 2005). Cathepsin B2 (TrCB2) was found as a transcript in the complementary DNA (cDNA) library of sporocysts with developing cercariae and by means of Western blotting in protein extracts of cercariae and schistosomula.…”