Cysteine proteinases from promastigotes of Leishmania (Viannia) braziliensis

Abstract

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.

Why strategies to control Leishmania spp. multiplication based on the use of proteinase inhibitors should consider multiple targets and not only a single enzyme

“…[18,[30][31][32][33][34] and Trypanosoma cruzi [35][36][37] and their importance in host-parasite interactions for over a decade. Collectively, these manuscripts make a relevant contribution to the search for proteinases that could serve as targets for developing future leishmaniasis treatments.…”

Affinity-Based Methods for the Separation of Parasite Proteins

“…This hypothesis is reinforced by reports indicating that lectin-glycan binding is important for the infection and virulence processes of some parasites, e.g. Acanthamoeba castellanii (Garate et al, 2006), H. contortus (Turner et al, 2008), L. (V.) braziliensis (Rebello et al, 2009) and T. gondii (Fauquenoy et al, 2008) …”

“…These enzymes are suggested to play a role in the process of parasite survival inside its hosts (Rebello et al, 2009). …”

Proteinases as virulence factors in Leishmania spp. infection in mammals

“…Within this context, our group has been working in an attempt to clarify the biological role of these enzymes. In a recent study, we were able to identify the cellular localization of CPs and their mechanism of anchoring to the parasite plasma membrane [84]. The organization of the cpb genes was also determined for this parasite species, along with the subsites of specificity for the recombinant CPB [85].…”