Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

Expression of metalloprotease activities during metacyclogenesis of a series of strains and clones of Trypanosoma cruzi was investigated using SDS-polyacrylamide gel electrophoresis with a range of different co-polymerized proteolytic substrates. A complex pattern of metalloprotease expression, with considerable quantitative and qualitative variability between strains and life-cycle stages, was apparent. This is in contrast to previously published data, and data presented in this study, which demonstrate a high degree of conservation of expression of cysteine proteases in different strains and clones of T. cruzi. All the metalloprotease activities identified partitioned into the detergent phase of a Triton X-114 extract, suggesting that they are membrane-bound. Developmental regulation of expression during metacyclogenesis, either in terms of quantity, or in terms of difference in relative amounts of different isoforms, was apparent for all isolates studied except CL14. However, a clearly metacyclic-specific/metacyclic-enriched metalloprotease was detected only in T. cruzi Dm28c and 383, and our results demonstrate that a metacyclic-specific metalloprotease common to all isolates of the parasite could not be detected, at least at this level of analysis.

To increase the existent genetic variability in cysteine proteases, a polymorphism study was performed in Haemonchus contortus by comparing 2 different strains of the parasite: North American (NA) and Spanish (SP) strains. For this purpose, the polymorphism of 5 previously reported genes (AC-1, AC-3, AC-4, AC-5 and GCP-7) were analysed by PCR–SSCP and sequencing procedures. Based on the SSCP results, a total of 20 different alleles were identified for the 5 loci assessed. Except locus AC-5, all the loci were polymorphic. Loci AC-1, AC-3, AC-4 and GCP-7 showed 5, 8, 2 and 4 alleles, respectively. The allelic frequencies ranged from 0·0070 to 0·8560 and were significantly different between strains. In addition, nucleotide diversity analyses showed a significant variation within and between strains. The variations in the nucleotide sequence of the different alleles were translated in some cases into changes in the amino acid sequence. Evidence of genetic variability in cysteine proteases from two different strains of H. contortus for the same set of genes had not been previously reported.