Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans

Abstract

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

Cleavage of cell junction proteins as a host invasion strategy in leptospirosis

“…To date, very few studies have been conducted experimentally to identify and characterize Leptospira l proteases (Dhandapani et al 2018 ; Thoduvayil et al 2020 ; Amamura et al 2017 ; Kumar et al 2022 ; Anu et al 2018 ; Sato and Coburn 2017 ; Martinez-Lopez et al 2010 ). To understand Leptospira l pathogenesis, it is mandatory to identify and characterize proteins mediating interactions with host components.…”