Production and characterization of the protease pappalysin from Leptospira interrogans

Pathogenic Leptospira species are extremely efficient in disseminating in the host, a fact attributed to their ability to escape complement system activation - the first line of defense of the innate immune response - and also to the ability to degrade extracellular matrix and other components of the human plasma. The secretion of proteases that inactivate host proteins is an important tool used by various microorganisms during the colonization process. The mechanisms underlying tissue damage caused by pathogenic leptospires are still poorly understood. Like other bacteria, leptospires cross epithelial and endothelial barriers to gain access to organs. Adhesion to and degradation of extracellular matrix components, especially the basement membrane, are certainly required for invasion. Recently, our group evaluated the proteolytic activity of secreted proteins by leptospires, and exoproteome analyzes of these bacteria allowed the identification of some proteases, including the metalloprotease pappalysin-1 domain protein, which we named leptolysin. In this work we produced and functionally characterized leptolysin from L. interrogans in order to expand our knowledge on this metalloprotease from Leptospira in the processes of invasion and immune evasion. According toin silico analyzes this protease belongs to the category of short pappalysins, also found in other bacteria. Leptolysin is present in all Leptospira species, but is more conserved among pathogenic species of the P1 subclade. A preliminary biochemical characterization of its proteolytic activity was performed using FRET (Free Resonance Energy Transfer) peptides. The enzyme exhibited maximum activity at pH 8.0 and 37oC, was active in the presence of different salts and was strongly inhibited by EDTA and 1,10-phenanthroline. It showed a marked preference for arginine residues in the P1 position. The proteolytic activity of recombinant leptolysin on host molecules was also evaluated in vitro and in vivo. The metalloprotease was active against extracellular matrix proteins (proteoglycans and fibronectin), coagulation cascade molecules (fibrinogen and thrombin) and effector proteins of the human complement system (C2 to C9). In mice dorsal skin leptolysin caused hemorrhages and degradation of skin fibronectin. With regard to the impacts produced by the protease on coagulation, human plasma treated with high doses of the protein failed to clot. Analyzes of the microcirculation by intravital microscopy (cremaster muscle model) showed an increase in the number of rolling leukocytes in the presence of leptolysin, and time-dependent leukocyte aggregation was observed. A leptolysin knockout strain (&#916lic13434) was produced and characterized. This strain showed lower survival in normal human serum (SHN) compared to the wild-type strain. However, in a model of epicutaneous infection in hamsters, no attenuation of virulence was observed with the knockout strain, although the bacterial load in the kidneys of these animals was lower than that observed in animals inoculated with the wild-type strain. Finally, data with sera from leptospirosis patients suggest that leptolysin is produced during natural infections by pathogenic leptospires. The characterization of toxins, their targets and mechanisms of action can help in the development of strategies to combat leptospirosis. (AU)