Understanding serine proteases implications on Leishmania spp lifecycle.

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment.

Reactivity of sera from dogs living in a leishmaniasis-endemic area to the COOH-terminal region of cysteine proteinase B.

Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n=114), including suspect (n=30) and positive (n=50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n=34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density=0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.

A ß-mercaptoethanol-modified enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis.

Two immunoglobulin G enzyme-linked immunosorbent assay (ELISA) versions using whole promastigotes of Leishmania infantum (syn. Leishmania chagasi) treated either with ß-mercaptoethanol (ß-ME-ELISA) or trypsin (TRYP-ELISA) as antigens were developed for the diagnosis of canine visceral leishmaniasis (CVL). By comparison with the direct agglutination test (DAT; 100%, 31/31; 95% confidence interval [CI]: 86.3-100%), slightly lower sensitivity was demonstrated for the newly developed ß-ME-ELISA (93.5%, 29/31; 95% CI: 77.2-98.9%). Sensitivity was higher for ß-ME-ELISA compared with TRYP-ELISA (87.1%, 27/31; 95% CI: 69.2-95.8%) in serum samples from dogs with CVL. When tested with sera from 37 healthy dogs and from 45 dogs with clinical conditions other than CVL, a specificity of 97.6% (80/82; 95% CI: 90.1-99.6%) was estimated for ß-ME-ELISA as compared to 100% (82/82; 95% CI: 94.4-100%) and 95.1% (78/82; 95% CI: 87.3-98.4%) for DAT and TRYP-ELISA, respectively. Observed agreement was 94.0% (95% CI: 88.7-97.1%) between DAT and ß-ME-ELISA (κ = 0.879; 95% CI: 0.803-0.956) and 87.4% (95% CI: 80.8-92.1%) between DAT and TRYP-ELISA (κ = 0.743; 95% CI: 0.636-0.851). Current results advocate application of the new ß-ME-ELISA for diagnosis of CVL at the laboratory level and confirmation of results obtained with the DAT in field studies.