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

Abstract

Leptospirosis is a zoonosis caused by spirochete bacteria that belong to the genus Leptospira. This disease represents a serious public health problem, especially in developing countries with tropical and subtropical temperatures. Pathogenic leptospires escape from the Complement System, a property that permits them to survive in vitro when in contact with normal human serum (NHS). In a previous study carried out by our group, it was observed that culture supernatants from different pathogenic species of leptospires (SPL) contain proteases that cleave many Complement proteins, including the central molecule C3 and its fragments C3b and iC3b. Our hypothesis is that these proteases, could decrease the phagocytic clearance of leptospires. Using flow cytometry, we observed decreased amounts of CR3 and CR4 in murine peritoneal macrophages treated with SPL for 24 h. By confocal microscopy, we observed reduction in TLR2, CD11b and CD206 levels when these cells were treated with SPL and recombinant thermolysin for 24 h. Furthermore, opsonins such as C3b/iC3b deposited on the surface of pathogenic leptospires were observed to be completely degraded in the presence of SPL or recombinant thermolysin. Finally, we decided to investigate the phagocytosis of pathogenic leptospires by macrophages in the presence of these proteases. We observed an increase of phagocytosis of leptospires opsonized with normal mouse serum even when macrophages were treated with the proteases. However, when opsonized bacteria were also incubated with SPL, recombinant thermolysin and recombinant leptolysin., there was a decline in leptospires phagocytosis. This suggests that the proteolytic activity can affect phagocytosis by peritoneal macrophages mainly through the degradation of opsonins deposited in the membrane of leptospires. These observations lead us to suggest that proteases secreted by pathogenic leptospires could degrade opsonins present in normal serum or deposited in 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.