Proteolytic Activity of Secreted Proteases from Pathogenic Leptospira on Phagocytosis by Murine and Human Macrophages

Author(s)

Thaís Akemi Amamura

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2022-01-21

Examining board members

Lourdes Isaac; Niels Olsen Saraiva Câmara; Fernanda Calheta Vieira Portaro; Elsio Augusto Wunder Junior

• Advisor: Lourdes Isaac

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 sub-tropical temperatures. Pathogenic leptospires escape from the Complement System, being able 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 several pathogenic species of leptospires (SPL) contain proteases that cleave many Complement proteins, such as, the central molecule C3, as well as its fragments C3b and iC3b. Our hypothesis is that such proteases, including thermolysin and pappalysin, could decrease the uptake and death of leptospires by phagocytes. Using flow cytometry, we observed lower presence of LFA-1, CR3 and CR4 in murine peritoneal macrophages treated with SPL for 24 h. By confocal microscopy, we observed less presence of TLR2, CD11b and CD206 when these cells were treated with SPL for 24 h. Human macrophages (THP-1) showed lower presence of TLR2, TLR4, CD206 and CR1 on their surface, after treatment with SPL and thermolysin. Furthermore, opsonins such as C3b/iC3b deposited on the surface of pathogenic leptospires can also be completely degraded in the presence of SPL or recombinant thermolysin. Finally, the phagocytosis of pathogenic leptospires by THP-1 significantly decreased when these cells were treated with recombinant SPL, thermolysin or pappalysin compared with cells treated only with PBS. Thus, this study suggests that proteases secreted by pathogenic leptospires could interfere with their own recognition and internalization by murine or human macrophages, by degrading opsonins present in NHS or deposited in the bacterial membrane as well as in the cleavage or inhibition of macrophage surface molecules, thus favoring the spread of leptospires in the organism. (AU)

• FAPESP's process: 17/10208-9 - Evaluation of the proteolytic activity of proteases secreted by pathogenic leptospires in phagocytosis by human and murine phagocytes
• Grantee: Thais Akemi Amamura
• Support Opportunities: Scholarships in Brazil - Doctorate