The role of amastins in inflammasome activation and stability of vacuoles infected by Leishmania braziliensis

Author(s)

Danilo Sasso Augusto

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-08-23

Examining board members

Dario Simões Zamboni; Munira Muhammad Abdel Baqui; Vanessa Carregaro Pereira; Santuza Maria Ribeiro Teixeira

• Advisor: Dario Simões Zamboni

Abstract

Leishmaniasis is a neglected tropical disease. Currently, about 12 million people are affected, suffering from ulcerated skin lesions to deaths. Part of the life cycle of Leishmania spp. parasites, the etiological agent of leishmaniasis, occurs in the digestive system of phlebotomine vector and part occurs in mammalian tissue, within the vacuoles of phagocytic cells. Recently, the inflammatory response of Nlrp3, a high molecular weight cytoplasmic cell complex related to the inflammatory response to damage and pathogens, was elucidated in infection by Leishmania spp. parasites, generating IL-1? and NO, which have microbicidal and inflammatory responses. With the validation of the intracellular machinery of RNAi in Leishmania braziliensis, it was possible to silence and study the role of amastins, a family of surface glycoproteins expressed in amastigotes of Leishmania spp. related to the adhesion between the amastigote membrane and the internal membrane of the parasitophores vacuoles. This work aims to understand the response of the inflammasome to an infection with L. braziliensis parasites with silenced amastins. Macrophages infected with parasites with silenced expression of amastins showed greater activation of caspase-1 and release of IL-1? dependent on the activation of Nlrp3 when compared to macrophages infected with wild parasites or with reconstituted amastine expression. Caspase-11-/- and Nlrc4 - /- macrophages showed reduction in total IL-1? production in L. braziliensis infections. However, such inflamomasomes are not directly involved in the specific increase of IL- 1? release in infections by parasites with irregular expression of amastins. Aiming to understand how the absence of amastin induces a greater activation of inflammasomes, an immunofluorescence of infected macrophages was performed using a galectin-3 specific antibody, a marker of vacuole lysis. These experiments elucidated the role of amastins on the maintenance of vacuolar stability, so that macrophages infected by parasites with amastins deficient expression presented a much larger number of vacuole lysis events when compared to macrophages infected with parasites with reconstituted expression of amastins. Finally, it was found that in L. braziliensis infection, galectin-3 plays a role in the enhancement of Nlrp3 activation regardless of the presence or absence of amastins. From the experiments it is possible to conclude that the amastins are correlated to the maintenance of the stability of the vacuole so that possibly molecular patterns associated with pathogens (PAMPs) are being released in the cytoplasm of macrophages infected by the strains with silenced expression of amastins. Possibly such PAMPs are recognized by receptors of innate immunity, inducing potassium efflux and consequent greater activation of Nlrp3 (AU)

• FAPESP's process: 17/04243-6 - Investigation of the role of amastins in the inflammasome activation and in the translocation of Leishmania molecules into the macrophage cytoplasm
• Grantee: Danilo Sasso Augusto
• Support Opportunities: Scholarships in Brazil - Master