Proteolytic activity of secreted proteases from pathogenic leptospires and effects on phagocytosis by murine macrophages

Abstract

Leptospirosis is a zoonosis caused by spirochete Leptospira. Pathogenic leptospires evade the Complement System, enabling their survival upon contact with normal human serum in vitro. In a previous study, we demonstrated that proteases secreted by pathogenic leptospires cleave several Complement proteins, including C3 and the opsonins C3b and iC3b. We hypothesize that these Leptospira proteases, such as thermolysin and leptolysin, may decrease the phagocytic activity of murine peritoneal macrophages. We observed decreased amounts of CR3 and CR4 using flow cytometry when these cells were treated with supernatant from the culture of pathogenic leptospires (SPL) for 24 h. Through confocal microscopy, we observed a reduction in TLR2, CD11b, and CD206 (mannose receptor) levels when these cells were treated with SPL or recombinant thermolysin for 24 h. Furthermore, opsonins such as C3b/iC3b deposited on the surface of pathogenic leptospires were clearly degraded in the presence of recombinant thermolysin or recombinant leptolysin. Consequently, when opsonized bacteria and macrophages were previously incubated with these proteases, phagocytic activity was diminished. These observations lead us to suggest that proteases secreted by pathogenic leptospires could degrade opsonins present in normal serum or deposited on the bacterial membrane, as well as cleave or inhibit macrophage surface molecules. Therefore, these proteases could interfere with the recognition and internalization by murine macrophages, favoring the spread of leptospires in the host.

Keywords: Complement system; Immune evasion; Leptospira; Macrophages; Phagocytosis; Proteases.