Dvořák J., Caffrey C.R., Horák P. (2002): Cysteine proteases of Trichobilharzia regenti. 11. Helmintologické dny, Dolní Věstonice, 13.-16. května 2002. In: Helminthologia 39, 3: 173.

ISSN 0440-6605,

IF 0,793

GACR 204/00/1095, GACR 524/00/0622, GAUK 123/2000/b-BIO/PřF, J13/981131-3, J13/981131-4

Members of the Schistosoma genus are parasites of vertebrates that ingest blood components as a source of nutrition. Trichobilharzia regenti, which resides in the nasal cavity of birds, is the only known species from the family Schistosomatidae to digest other host tissues. The larvae (schistosomula) of T. regenti migrate through the peripheral and central nervous system in order to reach the brain and, finally, the nasal area. This migration can cause neuromotor disorders and paralysis in bird hosts (ducks) and in experimentally infected mice. Nervous tissue serves as a nutrient source during migration and development, whereas, in nasal mucosa, adult flukes feed on blood. To date, there is no information regarding proteolytic enzymes (proteases) elaborated by T. regenti that might facilitate digestion of or migration through nervous tissue. Also, the presence of such proteases in T. regenti excretory-secretory products may modulate host immune responses. As Clan CA Family C1 (papain-like) cysteine proteases are involved in the digestion of blood components by schistosomes, these enzymes were chosen for this primary analysis of T. regenti.
Proteins from lyophilized 5-9 day old schistosomula (500, both sexes) were dissected from the spinal cord of infected laboratory ducks (Anas platyrhynchos), cleaned and pulse-sonicated in 200 µl 0.1 M buffer (citrate-phosphate or Bis-Tris, pH 6.0). After centrifugation 10 000 x g for 5 min, the supernatant was stored at -80 0C. Cathepsin B- and L-like protease activity in the supernatant was measured using 20 µM of the synthetic peptidyl fluorogenic substrates Z-Phe-Arg-NMec (cleaved by cathepsin B and L) and Z-Arg-Arg-NMec (selective only for cathepsin B) in the presence of 2 mM DTT. Controls contained water instead of supernatant. Inhibition of activity was accomplished with 10 µM E-64. Released NMec was quantified in a fluorometer (excitation 360 nm; emission 460 nm). Protease activity against Z-Phe-Arg-NMec was detected between pH 3.0 and 8.0 with a maximum at pH 5.0: activity against Z-Arg-Arg-NMec was only detected between pH 5.5 and 6.5 with a maximum at pH 6.0. Preference for Z-Phe-Arg-NMec over Z-Arg-Arg-NMec was 10-fold (at pH 6). Cysteine proteases in T. regenti extracts were identified by the use of Bodipy-green DCG-04 (a fluorescent analog of E-64 (Greenbaum 2000)), SDS-PAGE and fluorescence scanning densitometry. A predominant protease band of 33 kDa was labeled. Mono Q anion exchange chromatography partially isolated the 33 kDa protease. This protease is tentatively identified as an ortholog of schistosome cathepsin B, due to (1) its molecular mass, and (2), its specificity for both peptidyl substrates. However, this identification will be experimentally confirmed by sequence analysis. Further studies, including cloning and heterologous expression of the protease followed by detailed enzyme kinetics are planned.