Stage-Specific Pathways of Leishmania infantum chagasi Entry and Phagosome Maturation in Macrophages

Abstract

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival.

Bioanalysis of Eukaryotic Organelles

“…One study investigated the role of the sodium ion channel NaV1.5 on phagosome maturation in human peripheral blood monocytes, which were differentiated to macrophages and treated with dead bacillus Camille-Guerin, a model for mycobacterial infection. 452a Confocal immunofluorescence microscopy showed that NaV1.5 was localized in the endosomes near the periphagosome region. Knockdown of NaV1.5 caused about a 9-fold decrease in phagocytosis of the dead bacillus.…”

“…451 Next, phagosome-lysosome interactions with pathogen-based models 452 and phagosome-autophagosome interactions are discussed. 453 Finally we discuss recent insights into phagosome interactions with other membrane systems such as the plasma membrane 454 , ER, 455 and Golgi.…”

“…The role of Rab14 in phagosome maturation was studied with M. tuberculosis -infected mouse macrophage cells and human embryonic kidney cells. 452b Although Rab14 is known to participate in early endosome-Golgi trafficking, confocal fluorescence microscopy revealed that Rab 14 was colocalized with phagosomes containing M. tuberculosis . Decreased expression of Rab14 with siRNA knockdown caused increased interaction of lysosomes and phagosomes containing M. tuberculosis .…”

“…A recent study investigated the role of phagosome membrane permeability on the fusion of the phagosome with lysosomes in L. monocytogenes -infected mouse macrophages. 452c Confocal fluorescence microscopy was used to monitor the pH and calcium concentrations in phagosomes using ratiometric fluorescent probes. In cells expressing listeriolysin O, phagosomes were permeabilized, and the pH and calcium concentration of the L. monocytogenes -containing vacuoles remained close to cytosolic pH, i ndicating compromised membrane permeability.…”

Interaction of pathogens with host cholesterol metabolism

“…Mycobacterium tuberculosis uses lipid rafts to enter alveolar epithelium and it was suggested that it secretes a factor causing rafts aggregation to prepare uninfected cells for new infection [21]. Leishmania donovani also binds to lipid rafts of the macrophages using them as an entry gate; cholesterol depletion inhibits infection in vitro [22]. …”