Identification and characterization of nuclease activity in extracellular Leptospiral lipoprotein, LipL41

Bacterial lipoproteins are outer surface proteins usually anchored to a cell's membrane by an N-terminally linked lipobox. Pathogenic Leptospira interrogans expresses numerous extracellular lipoproteins on its outer surface. Many lipoproteins are involved in host-pathogen interactions and are identified as potential vaccines and diagnostic candidates. At the same time, their role in hydrolytic activity has not been thoroughly investigated. In this study, we examined and identified a functional nuclease motif in extracellular Leptospiral lipoprotein LipL41. Notably, LipL41 was found to contain C-terminal TPR (tetratricopeptide) repeats and a putative nuclease motif overlapping with the TPR. The recombinant LipL41 demonstrated DNA cleavage activity on different types of DNA, while the C-terminal truncated form, LipL41Δc40, did not exhibit DNA cleavage. Moreover, LipL41 possessed robust DNA cleavage activity over a wide pH spectrum, highlighting its potential functional stability and adaptability under varying physiological conditions. The electrostatic surface analysis of LipL41 indicated that the positively charged region was primarily composed of Lysine residues. Molecular docking and molecular dynamics simulation revealed a stable binding of the protein to a DNA strand. The possession of nuclease activity in LipL41 may physiologically facilitate the hydrolysis of DNA content within the host's neutrophil extracellular trap (NET). To our knowledge, this is the first report highlighting the nuclease activity in LipL41. These findings significantly enhance our understanding of the functional diversity among extracellular lipoproteins.