The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µm (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

In the present paper, we have analysed the cellular and extracellular proteolytic activity profiles in 2 distinct Leishmania braziliensis strains: a recently isolated (virulent) and a laboratory-adapted (avirulent) strain. Quantitative and qualitative differences on the peptidase expression were observed in both strains. For instance, low-molecular mass acidic cysteine peptidase activities were detected exclusively in the virulent strain. Similarly, metallopeptidase activities were mainly produced by L. braziliensis virulent promastigotes. Interestingly, metallo- and cysteine peptidase activities were drastically reduced after several in vitro passages of the virulent strain. Western blotting, flow cytometry and fluorescence microscopy analyses were performed to detect homologous of the major leishmania metallopeptidase (gp63) and cysteine peptidase (cpb) in virulent and avirulent strains of L. braziliensis. Our results revealed that the virulent strain produced higher amounts of gp63 and cpb molecules, detected both in the surface and cytoplasm regions, than the avirulent counterpart. Metallo- (1,10-phenanthroline and EGTA) and cysteine peptidase (E-64) inhibitors arrested the growth of L. braziliensis virulent strain in a dose-dependent manner, as well as the association index with peritoneal murine macrophages. Conversely, these peptidase inhibitors did not affect either the proliferation or the cellular interaction of the avirulent strain. Corroborating these findings, the pre-treatment of the virulent strain with both anti-peptidase antibodies promoted a prominent reduction in the interaction with macrophages, while the association index of the avirulent strain to macrophage was only slightly diminished. Moreover, the spent culture medium from virulent strain significantly enhanced the association index between avirulent strain and macrophages, and this effect was reversed by 1,10-phenanthroline. Collectively, the results presented herein suggest that peptidases participate in several crucial processes of L. braziliensis.

Excised axes from cotton (Gossypium hirsutum L. cv. M-8, a double haploid) were imbibed in vitro for 3,6,9,12,18 and 24 h. Data for length, fresh and dry weight and percentage water showed that excised axes undergo a triphasic pattern of growth: an initial burst, a short lag phase and then protracted rapid growth. Analysis of protein content with SDS-PAGE, and of activities of amino-, carboxy- and endopeptidases provided data indicating a direct correlation between major axis elongation growth between 12 and 24 h and enzymatic breakdown of storage proteins (especially those of 48 kDa) by carboxy- and endopeptidase. Data for axes imbibed at 30°C and 0–5°C for 12, 18 and 24 h indicated that a reduction in length and percentage moisture in the cold occurred in parallel with reductions in carboxy- and endopeptidase activities but not in ami-nopeptidase activity. The tentative conclusion is that carboxy- and endopeptidase are probably synthesized de novo in the axis during imbibition and are important in initial breakdown of storage proteins for axial elongation growth.