Heterologous Expression, Purification, and Characterization of a High-Temperature Requirement A (HtrA) Protease...

Leptospirosis is a zoonotic disease affecting humans in the tropical and temperate regions. Considerably high mortality rate (60 per 1000 adult) and associated morbidity necessitate the need for efficient diagnostic and therapeutic approaches for this disease. Proteins that play crucial roles in the invasion/pathogenesis are potential candidates for the diagnosis/therapeutics. High temperature requirement A (HtrA) is a protein expressed by many pathogenic bacteria, important for their virulence and survival. In this study, we have amplified, cloned, and expressed one of the HtrA homologues (HtrA1) from Leptospira. The expressed recombinant HtrA was purified using Ni-NTA chromatography. Physicochemical characterization of the enzyme using azo-casein substrate showed the maximum activity at a temperature 42 °C and pH 7. While Mn²⁺ showed significant positive effect, all the other tested metals inhibited the enzymatic activity, sometimes up to 97% as in the case of Cu²⁺. All the protease inhibitors inhibited the enzymatic activity with PMSF having maximum efficiency. The host cells expressing HtrA showed growth inhibition in a time-bound manner. Docking analysis identified the crucial amino acids involved in the interaction with cell junction proteins like E-cadherin, occludin, claudin-8, and desmoglein-2. Treating adherent mammalian cells with the recombinant protein showed the disruption of cell adherence, and the western analysis of the protein samples collected from the same experiment indicated the cell junction protein cleavage when probed with anti-E-cadherin antibody.