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Receptor-Mediated Phagocytosis and Parasitophorous Vacuole Maturation of Leishmania Amazonensis Promastigotes

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Master's thesis

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Leishmania parasites gain access to their intracellular niche through phagocytosis. Phagocytosis is a receptor-mediated process that has been shown to involve the use of opsonin dependent as well as opsonin independent receptors. This study ascertains the importance of the opsonin dependant receptors CR3 and FcyR to the cellular entry of Leishmania amazonensis. Furthermore, it assesses differences in phagosome composition and maturation as a result of internalization through these receptors. RAW 264.7 macrophages were infected with non-opsonized, complement opsonized, or anti-parasite antibody opsonized L. amazonensis promastigotes. The composition of parasite-containing phagosomes was monitored by immunofluorescence microscopy up to 480 min post-infection. Promastigotes firmly established infection by 30 min and were 40 more efficient at cellular entry when opsonized with complement, 13 more efficient when antibody opsonized. By 5 min post-infection, 88 of internalized complement opsonized parasites were in vacuoles transiently reactive with anti-CR3 antibody and most exhibited characteristic association with vinculin. In contrast, only 29 of antibody opsonized parasite vacuoles were anti-CR3 reactive and 68 were positive for vinculin. LAMP1 colocalization steadily increased throughout the infection under all opsonization conditions and nearly all phagosomes contained this molecule by 75 min post-infection. Rab5a association persisted with approximately 40 of phagosomes harboring complement and non-opsonized parasites for at least 90 min, compared to rab7, which was present in negligible amounts throughout the experiments. In contrast, antibody opsonized promastigotes showed approximately 9 rab5a association and 5 rab7 association with similar kinetics. Cathepsin S was detected in association with phagosomes without the involvement of CI-M6PR.

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  • Biology
  • Microbiology

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