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

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.

Serine Proteases Profiles of Leishmania (Viannia) Braziliensis Clinical Isolates With Distinct Susceptibilities to Antimony

“…The serine protease activity measured by Z-FR-AMC, a substrate for serine proteases such as cathepsins, kallikrein and plasmin (41), was relatively the same among promastigotes and axenic amastigotes. Serine enzymatic activity is generally con rmed by using PMSF, AEBSF and TLCK inhibitors (26). Besides, PMSF and AEBSF are known to inhibit a broad range of serine proteases including subtilisins while TLCK has a greater preference for Leishmania spp.…”

“…Additionally, based on the previous role seen for subtilisins as maturases of TXNPx (18,26), our study examined the expression of subtilisin transcripts from SB clan and S8 family (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080). In general, almost all isolates (promastigotes and axenic amastigotes) expressed the three mentioned genes.…”

“…The primers used in this study were previously designed and used to detect serine proteases and tryparedoxin peroxidase transcripts (26). Brie y, the design is based on the L. (V.) braziliensis subtilisins (LbrM.…”

Insights into Leishmania Molecules and Their Potential Contribution to the Virulence of the Parasite

“…Recently, it was reported that L. (Viannia) braziliensis promastigotes express serine proteinases that have distinct subcellular distributions and expression. Taken together, this might contribute to the maintenance of this parasite’s lifestyle at physiological pH, in the cytosol and on the external face of the parasitic membrane [ 134 ]. In stark contrast, the proteinases secreted from the host also affect the dynamics and progression of the infection, in addition to the development of the lesion [ 135 ].…”

“…In this concern, OPB facilitates the establishment of the infection of murine macrophages by L. (L.) donovani [ 144 ], while it is associated with maintaining the infection of murine macrophage in L. (L.) major [ 145 ]. A recent study proposed that promastigotes of L. (V.) braziliensis express SPs that contribute to the maintenance of this parasite’s lifestyle at physiological pH and the out membrane of the parasite [ 134 ]. Taken together, odorant-binding protein (OBP) regulates the degradation of enolase–plasminogen complexes of the parasite, immune evasion, and disease pathology [ 144 ].…”