Trichomonas vaginalis induces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose on T. vaginalis cytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface of T. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.

Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5–24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.

Eight strains of mice, of contrasting genotypes, infected with Heligmosomoides bakeri were studied to determine whether the anthelmintic efficacy of papaya latex varied between inbred mouse strains and therefore whether there is an underlying genetic influence on the effectiveness of removing the intestinal nematode. Infected mice were treated with 330 nmol of crude papaya latex or with 240 nmol of papaya latex supernatant (PLS). Wide variation of response between different mouse strains was detected. Treatment was most effective in C3H (90·5–99·3% reduction in worm counts) and least effective in CD1 and BALB/c strains (36·0 and 40·5%, respectively). Cimetidine treatment did not improve anthelmintic efficacy of PLS in a poor drug responder mouse strain. Trypsin activity, pH and PLS activity did not differ significantly along the length of the gastro-intestinal (GI) tract between poor (BALB/c) and high (C3H) drug responder mouse strains. Our data indicate that there is a genetic component explaining between-mouse variation in the efficacy of a standard dose of PLS in removing worms, and therefore warrant some caution in developing this therapy for wider scale use in the livestock industry, and even in human medicine.

Invasive properties of pathogenic Entamoeba histolytica have been postulated to depend on the secretion or release of cysteine proteinases and pore-forming peptides (amoebapores) by trophozoites. To establish whether such toxic molecules are released by viable trophozoites or upon cellular disintegration, amoebae were maintained in various culture media, and activities in supernatants were monitored over time in correlation to cellular integrity. By measuring the release of the cytoplasmic marker enzyme NADP+-alcohol dehydrogenase, it became apparent that release of amoebapore was accompanied by cellular disintegration. In contrast, considerable quantities of cysteine proteinases were found to be present in culture supernatants also when amoebae remained intact. Treatment of amoebae with concanavalin A, bacterial lipo-polysaccharides or the calcium ionophore A23187 did not result in amoebapore secretion suggesting that here target cell contact is required as an essential stimulus.

The excreted/secreted proteinases of Haplometra cylindracea maintained in vitro, were found to hydrolyse the fluorogenic substrates, Z-ArgArg-NHMec and Z-PheArg-NHMec. This activity was shown to be typically that of cysteine proteinases, as turn-over of both substrates could be blocked by pre-incubation with peptidyl diazomethyl ketones. The biotinylated affinity reagent, biotin-Phe Ala-DMK, used in combination with Z-PheTyr(OBut)-DMK, was employed for the labelling and characterization of these cysteine proteinase activities. Three cathepsin B-like species were detected, with molecular weights of 48, 22–23 and 14 kDa, together with a cathepsin L-like enzyme, with a molecular weight of 55 kDa. The proteinases were also found to have hydrolytic activity towards the substrate, Z-GlyGlyArg-NHMec, which could be blocked by pre-incubation with either of the serine proteinase-selective reagents, Z-ArgP(OPh)2, or biotin-LysP(OPh)2, showing the activity to be trypsin-like. Using the biotinylated affinity label to characterize the trypsin-like enzymes revealed two molecular species with molecular weights of 20 and 24 kDa.

Intracellular differentiation of Leishmania promastigotes to amastigotes is a critical step in the establishment of infection. In this report three related features of mexicana subspecies amastigotes were used to follow the differentiation of the parasites within macrophages. Early after infection, (a) parasites did not contain ultrastructurally recognizable megasomes, (b) cysteine proteinase activity of parasite lysates was not detected in gelatin-containing acrylamide gels, and (c) parasites were essentially resistant to L-leucine-methyl ester (Leu-OMe). Typical megasomes were first identified on the 5th day, were more prevalent on day 7, and underwent swelling in macrophages exposed to Leu-OMe. Cysteine proteinase activity was first detected on day 3 and increased thereafter. Susceptibility to Leu-OMe of parasites studied in situ or isolated from infected macrophages increased with time of intracellular residence and by 7 days approached that of amastigotes isolated from mouse lesions. In contrast, parasites derived from either promastigotes or amastigotes were equally susceptible to another leishmanicidal compound, tryptophanamide (Trp-NH2). The results provide additional support for the involvement of megasomes and their cysteine proteinases in parasite killing by Leu-OMe, and highlight the slow pace of the intracellular differentiation of L. amazonensis promastigotes to amastigotes.

The haemolytic activity of live Trichomonas vaginalis organisms was investigated. Optimal haemolysis of human erythrocytes was observed at a parasite to erythrocyte ratio of 1:5 during a 2 h incubation period. No haemolytic activity was detected in concentrated culture supernatants after overnight growth of trichomonads or when parasites were separated from erythrocytes by a 3 μm filter, suggesting a contact-dependent mechanism for haemolysis. The haemolytic activity was temperature-dependent and maximal haemolysis occurred at 37 °C. Treatment of trichomonads with metronidazole reduced levels of haemolysis by > 50%. Maximal haemolysis occurred at the pH range of the vagina during trichomoniasis. N-μ-tosyl-L-lysyl-chloromethyl ketone and iodoacetamide, inhibitors of trichomonad cysteine proteinases, reduced the haemolytic activity of live parasites.

Cell extracts of an entero-invasive protozoon of squirrel monkeys, Tritrichomonas mobilensis, contained relatively high proteolytic activity, measured on hide powder azure (HPA). Multiple proteinase forms, optimally active at pH 5–7, were detected by electrophoretic analysis in gelatin-containing polyacrylamide gels. Three major proteinase bands of apparent low molecular weights, Mr 18, 23 and 30 kDa, were seen on gels. Inhibition-activation studies suggest that only cysteine proteinases were involved in HPAase and gelatinolytic activities of T. mobilensis cell extracts.

With the aim to study proteinases released to the culture medium during Trypanosoma cruzi metacyclogenesis, the presence of cysteine proteinases (CPs) was analysed in culture supernatants obtained throughout the differentiation induced by stimulation of epimastigotes with Triatoma infestans hindgut homogenate. In SDS-gelatin containing gels, an important endopeptidase activity with apparent molecular weight range between 97 and 116 kDa was encountered at pH 6, which was abolished by the specific cysteine proteinase inhibitor E-64 and TLCK, but not by pepstatin, 1,10 phenantroline or PMSF. This novel CP, named TcCPmet, showed affinity to cystatin-Sepharose, denoting its thiol-proteinase character as well as to ConA-Sepharose, indicating it contains N-linked oligosaccharides. However, it presented a different elution pattern on ConA-Sepharose than cruzipain and, in addition, it was not recognized by anti-cruzipain serum, facts that strongly suggest the different nature of both CPs. Moroever, evidence is presented indicating that TcCPmet was able to hydrolyse the same chromogenic peptides as cruzipain at optimal alkaline pH values, although with a different order of effectiveness. Our results indicate the presence of a novel CP secreted by metacyclic trypomastigotes and reinforces the important role of these enzymes in metacyclogenesis.

The expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants. We have comparatively analysed the proteolytic profiles of 19 monoxenous trypanosomatids (Herpetomonas anglusteri, H. samuelpessoai, H. mariadeanei, H. roitmani, H. muscarum ingenoplastis, H. muscarum muscarum, H. megaseliae, H. dendoderi, Herpetomoas sp., Crithidia oncopelti, C. deanei, C. acanthocephali, C. harmosa, C. fasciculata, C. guilhermei, C. luciliae, Blastocrithidia culicis, Leptomonas samueli and Lept. seymouri) and 4 heteroxenous flagellates (Phytomonas serpens, P. mcgheei, Trypanosoma cruzi and Leishmania amazonensis) by in situ detection of enzyme activities on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE ) containing co-polymerized gelatine as substrate, in association with specific proteinase inhibitors. All 23 trypanosomatids expressed at least 1 acidic proteolytic enzyme. In addition, a characteristic and specific pattern of cell-associated metallo and/or cysteine proteinases was observed, except for the similar profiles detected in 2 Herpetomonas (H. anglusteri and H. samuelpessoai) and 3 Crithidia (C. fasciculata, C. guilhermei and C. luciliae) species. However, these flagellates released distinct secretory proteinase profiles into the extracellular medium. These findings strongly suggest that the association of cellular and secretory proteinase pattern could represent a useful marker to help trypanosomatid identification.

Axenic trophozoites of Entamoeba histolytica strain HM1-IMSS grown in vitro in the presence of E-64, a potent irreversible inhibitor of cysteine proteinases, preserved their viability, their rate of replication, their resistance to complement, their haemolytic capacity and their ability to destroy target cells, despite complete inhibition of total cysteine proteinase activity. On the other hand, their erythrophagocytic capacity and their ability to decrease TER of MDCK cells was partially decreased. The same trophozoites injected into the portal vein of hamsters receiving a maintaining dose of E-64 failed to cause tissue damage and were rapidly eliminated. Our results suggest that amoebic cysteine proteinase activity is not required for amoebic functions in in vitro conditions, but that it becomes necessary for survival of trophozoites in in vivo conditions, whatever other role (if any) it may play in the parasite's virulence.