Characterization of novel nuclease and protease activities among Leptospiral immunoglobulin-like proteins

Abstract

Bacterial immunoglobulin-like (BIg) domain containing proteins play a variety of biological functions. Leptospiral Immunoglobulin-like (Lig) proteins are well-known virulence factors located on the surface of the pathogenic Leptospira that act during adhesion, invasion, and immune evasion. The Lig proteins have many roles and have been designated as multifaceted proteins. However, the hydrolyzing function of Lig proteins is not yet investigated in detail. Here, we report novel in-vitro nuclease and protease activities in the Ig-like domain of LigA protein. All Ig-like domains were able to cleave DNA in the presence of a divalent ion, but not RNA. Site-directed mutagenesis revealed Mg⁺² binding residues in the Ig-like domain of LigA7. The basis of novel nuclease activity may be associated with protein adopting different conformation in the presence of divalent ions and substrate as investigated by change of intrinsic fluorescence. The docking of a stretch of double-strand DNA shows the binding on the positive surface of the protein. In addition, the protein is also observed to cleave a general protease substrate, β-casein, in our experimental condition. Our results proposed that the novel functions may be associated with neutrophil extracellular Trap (NET) evasion. Overall this study enhances the basic knowledge of non-nuclease proteins involved in the DNA cleavage activity and makes the foundation to explore its in-vivo activity in pathogenic Leptospira and other pathogens as well. Moreover, this information may be utilized to develop preventive strategies to interfere with Leptospira immune evasion.

Keywords: Immunoglobulin-like protein; Leptospira; LigA; Nuclease; Protease.