Proteínas relacionadas ao citoesqueleto e vias de sinalização envolvidas no tráfego intracelular de Leishmania amazonensis: efeito da proteína P21-His6 de Trypanosoma cruzi em infecção por Leishmania amazonensis in vivo

Abstract

CHAPTER I: Leishmania spp are obligate intracellular protozoan that can cause a complex of known infectious diseases such as leishmaniasis, affecting millions of people worldwide. The host-pathogen interaction involves the parasite surface molecules and cellular receptors that culminate in phagocytosis. This internalization process involves changes in cytoskeleton through interaction with proteins related to actin polymerization, as AFAP, Arp 2/3 complex, Galectin-3, Septins and WASp, as well as activation and signaling pathways. It is known that L. amazonensis promastigotes after entry into phagocytes are present in phagosomes, and these vesicles undergo a maturation process, forming phagolysosome, important for differentiation of the promastigote forms to amastigotes. This study aimed to understand which proteins related to actin cytoskeleton exert role in the process of invasion and multiplication of L. amazonensis and which signaling pathways are involved in the formation of phagolysosome, the invasion and multiplication of this protozoan process the host cell. For this, we did invasion and multiplication assays with macrophages C57 BL/6 and BALB/c peritoneal treated or not with cytochalasin D, invasion assay with macrophages C57 BL/6 previously fixed in formaldehyde (0.01%), assay with macrophages C57 BL/6 knockdown for AFAP-1L1, Arp2, Galectin-3, Septin 4, 14 and WASP proteins compared to wild type cells. And yet, assays phagolysosome kinetics of formation and proliferation and invasion assays using inhibitors of Akt, ERK2, MEK1, MEK1/2, mTOR, PI3K and Ras signaling pathways. The results show that L. amazonensis invaded cells defective in actin polymerization, as well as fixed cells as well as decreased expression of Arp2, Galectin-3 and WASp proteins increased internalization of these parasites. The signaling pathways MEK1/2, ERK2 and AKT delayed the recruitment of the lysosomes to phagosome, and inhibition of PI3K, MEK1/2 and ERK2 pathways drastically decreased internalization of the parasite in the cells. We concluded that L. amazonensis were able to actively invade phagocytic cells, and that the Arp2, Galectin-3 and WASp proteins are involved in the process of entry into host cells. In addition, the MEK1/2, AKT and ERK2 signaling pathways are involved in the formation of phagolysosome as well as PI3K, MEK1/2 and ERK2 pathways seem to favor infection with L. amazonensis. CHAPTER II: The flagellate protozoan Trypanosoma cruzi is the causative agent for Chagas disease. It is estimated that there are eight million people infected worldwide. T. cruzi has different surface proteins related to cell invasion. In this context, our research group identified in T. cruzi, a protein of 21 kDa (P21) showed that pro-phagocytic and chemotactic activities, playing an important role in the internalization of this parasite in vitro. This study aimed to evaluate the role of recombinant protein P21-His6 based on native P21 of T. cruzi in Leishmania amazonensis in order to seek greater understanding of the mechanism of action of this protein and its relationship with the host in vivo. To this end, the footpad infect BALB/c mouse treated with 40μg of active and denatured recombinant P21, BSA and PBS for 6 weeks and assess the footpad size, parasitic load (real time PCR) and cytokine production of IL-1β in the paws, the popliteal lymph nodes and spleen. The results showed an increase in the footpad area, as well as increased parasite load in the infected and treated animals with P21-His6. Also, increased production of IL-β in the footpad and the popliteal lymph nodes also in animals treated with P21-His6. We conclude that the P21 protein plays a role in pro-phagocytic also in vivo experiments, favoring infection.