Abstract
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.
Highlights
Leishmaniases are endemic diseases that are widespread in regions such as Asia, Europe, Africa, and the Americas
Reagents—Detergents [Tween 20, Triton X-100 (TX-100), and 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS)], gelatin, bovine serum albumin (BSA), penicillin, proteinase inhibitors [trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), phenylmethanesulfonyl fluoride (PMSF) and aprotinin], fluorogenic peptide substrates [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); ε= 1.78 × 104 M−1 cm−1], a ProteoSilverTM Silver Stain Kit, and Nancy-520 DNA gel stain were purchased from Sigma Aldrich Chemical Co
The results presented here bring new insight into the serine proteinases of L. (V.) braziliensis promastigotes and provide evidence of the active production of transcripts for both subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (TXNPx) (LbrM.15.1080) genes
Summary
Leishmaniases are endemic diseases that are widespread in regions such as Asia, Europe, Africa, and the Americas. Among the 53 Leishmania species reported, only 20 can cause human infection [2]. Parasite transmission occurs in a vector-host cycle through blood repast of female phlebotomines in the Lutzomyia or Phlebotomus genus in a vertebrate host [3]. The parasites are phagocytosed by macrophages and differentiate into amastigotes that, after intense multiplication by binary fission, rupture the cells and immediately infect new cells [4]. The appearance of ulcers may occur after the second week of infection [5,6], and after this initial lesion, the progression of the disease depends on several factors, including the Leishmania species and the patient’s immune system status. There are traditionally four clinical presentations of the disease: cutaneous leishmaniasis, cutaneous-diffuse leishmaniasis, cutaneous-mucosal leishmaniasis, and visceral leishmaniasis [1]
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