Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotes.

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not (P < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways.

Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process.

Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.

Microbial entry through caveolae: variations on a theme.

Caveolae and lipid rafts are increasingly being recognized as a significant portal of entry into host cells for a wide variety of pathogenic microorganisms. Entry through this mechanism appears to afford the microbes protection from degradation in lysosomes, though the level to which each microbe actively participates in avoiding lysosomal fusion may vary. Other possible variations in microbial entry through caveolae or lipid rafts may include (i) the destination of trafficking after entry and (ii) how actively the microbe contributes to the caveolae lipid/raft mediated entry. It seems that, though a wide variety of microorganisms are capable of utilizing caveolae/lipid rafts in various stages of their intracellular lifestyle, there can be distinct differences in how each microbe interacts with these structures. By studying these variations, we may learn more about the normal functioning of these cellular microdomains, and perhaps of more immediate importance, how to incorporate the use of these structures into the treatment of both infectious and non-infectious disease.

Caveolae as portals of entry for microbes.

Many pathogens, including many traditionally extracellular microbes, now appear capable of entry into host cells with limited loss of viability. A portal of entry shared by some bacteria, bacterial toxins, viruses and parasites are caveolae (or lipid rafts), which are involved in the import and intracellular translocation of macromolecules in host cells. A requirement for caveolae-mediated endocytosis of microbes appears to be that the respective receptor is a constituent of caveolae or must move to caveolae following ligation.