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.

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression.

Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 252 ± 20 µmol.min-1 mg of protein-1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min-1 mg of protein-1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.

Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance.

Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.

Increasing in cysteine proteinase B expression and enzymatic activity during in vitro differentiation of Leishmania (Viannia) braziliensis: First evidence of modulation during morphological transition.

Leishmania (Viannia) braziliensis presents adaptive protease-dependent mechanisms, as cysteine proteinases B (CPB). This study investigates the expression of three cpb gene isoforms and CPB enzymatic activity during the parasite differentiation. Relative expression levels of LbrM.08.0810 gene were assessed, exhibiting a higher quantity of transcripts in the logarithmic promastigotes phase than in the stationary promastigotes phase (>1.5 times). The cbp gene tends to decrease during acid pH shock and increases when the temperature rises (>1.3 times). LbrM.08.0820 and LbrM.08.0830 genes exhibited similar expression profiles to LbrM.08.0810 gene, with lower levels being observed overall. The proteolytic activity exhibits a gradual increase during the parasite's differentiation with low levels in samples of logarithmic promastigotes phase (3.2 ± 0.08 mmol min-1 mg protein-1) to a peak of activity after 72 h of incubation at 32 °C (4.2 ± 0.026 mmol min-1 mg protein-1) followed by a subsequent decrease of 68 % of peak activity levels after 96 h of incubation at 32 °C (2.8 ± 0.37 mmol min-1 mg protein-1). These activities were also measured in the presence of selective inhibitors for cysteine proteinases, such as Z-Phe-Phe-fluoromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, demonstrating their source as cathepsin-like proteinases. To the best of our knowledge, this report presents the first description of a modulation of cathepsin L-like expression during the L. (V.) braziliensis in vitro differentiation induced by acid pH and high temperature.

Proteinase inhibitors: a promising drug class for treating leishmaniasis.

This review presents and discusses the current status and perspectives of leishmaniasis treatment, with a special focus on the use of proteinase inhibitors. The history of treatment development, the first- and second-choice modern drugs and the advantages and disadvantages of using proteinases inhibitors as leishmanicidal treatments are presented and discussed. The reports gathered herein confirm the potential usefulness of proteinases inhibitors as an alternative or complement to the current leishmaniasis treatments. They also support the hypothesis that a combined treatment with multiple proteinase inhibitors may be efficient against Leishmania infections in vertebrate hosts.