In vitroselection ofPhytomonas serpenscells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

Abstract

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 µ m (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.

PDX proteins from Arabidopsis thaliana as novel substrates of cathepsin B: implications for vitamin B6 biosynthesis regulation

“…2A). Since Arabidopsis expresses both types of cysteine proteases [61,62], we decided to test additional inhibitors that are more specific for either the UPP (bortezomib [63]), cysteine proteases including calpains and cathepsins (MDL‐28170 [64,65]), or mainly cathepsin Bs (CA‐074 [66]) (Fig. 2A).…”

The Diverse Calpain Family in Trypanosomatidae: Functional Proteins Devoid of Proteolytic Activity?

“…To further explore the possible roles of P. serpens calpains, an MDL-resistant strain was generated and compared to the wild type cells. The major difference observed between both strains was the presence of microvesicles within the flagellar pocket of the resistant parasites, as revealed by ultrastructural analysis [28]. Table 1 summarizes the main effects of calpain inhibitors against trypanosomatid parasites reported by our research group.…”

Simvastatin Resistance of Leishmania amazonensis Induces Sterol Remodeling and Cross-Resistance to Sterol Pathway and Serine Protease Inhibitors

“…The efflux of the fluorescent probe Rhod-123 (Sigma-Aldrich Inc., St. Louis, MO, USA) was tested in the LaWT and LaSimR strains using a cytometer (CytoFLEX S Beckman Coulter). First, promastigotes of L. amazonensis (5 × 10 6 parasites/mL) in the log-phase of growth were incubated in RPMI medium in the presence and the absence of 100 µM of verapamil hydrochloride (Vp) (Sigma-Aldrich Inc., St. Louis, MO, USA), an inhibitor of the drug efflux pump P-glycoprotein, for 1 h at 26 • C. Next, parasites were incubated in the presence or absence of Rhodamine-123 (Rhod-123) (5 µg/mL) for 30 min [35,36]. The parasites were then washed three times, resuspended in 1 mL PBS buffer, and incubated in the presence or absence of Vp (100 µM) for 30 min and 90 min, in triplicates, to measure Rhod-123 efflux.…”