Infecção de células não fagocíticas por amastigotas de Leishmania amazonensis: um estudo dos mecanismos celulares envolvidos na invasão da célula hospedeira

Abstract

Intracellular parasites are characterized by the need to invade and live within host cells. For Leishmania spp. it is commonly assumed that the parasite must be captured by phagocytic cells, notably macrophages. However, several studies have described the presence of Leishmania spp. amastigotes within non-phagocytic cells. Since cell entry is a key process in infections by intracellular parasites we decided to investigate the mechanisms that allow Leishmania amazonensis amastigotes to invade cells lacking the ability to perform classical phagocytosis. Here we used different cell biology techniques to study the cell invasion process of non-phagocytic cells by L. amazonensis amastigotes in mouse embryonic fibroblasts (MEF). We performed and quantified infections, labelled infected cells and parasites to visualize molecules, organelles and structures possibly involved in cell invasion and proceeded analysis by different microscopy techniques. Our results show that living, but not PFA-fixed amastigotes, are rapidly internalized in MEFs, where they survive and persist within parasitophorous vacuoles rich in lysosomal markers. By labeling host cells with F-actin probes and using inhibitors of actin polymerization we demonstrated that invasion is dependent on host cell cytoskeleton. Confocal microscopies of early moments of infection indicate that the parasite is able to induce an extremely localized re-arrangement of host cell cytoskeleton to induce invasion. Since we had previously demonstrated that promastigotes use host cell lysosomes and not F-actin to invade MEFs we now propose that cell invasion of nonphagocytic cells by L. amazonensis is mechanistically different for each of the two evolutive forms presented by the parasite. The presence of Leishmania spp. amastigotes within non-phagocytic cells is an overlooked aspect of their biology. Studies on these new infection routes and on the role of non-phagocytic cells in the course of infection may provide important information about the biology of these parasites and the diseases they cause.