The virulence of the opportunistic fungal pathogen Candida albicans is in part due to its ability to switch between a yeast and hyphal form; permitting physical rupture and escape from macrophages after phagocytosis. This interesting feature makes C. albicans a good model organism to study in the context of phagosome maturation and in particular, the role of the small GTPase Rab14 in this process. Rab14 is recruited to bacterial phagosomes containing Chlamydia trachomatis (Capmany & Damiani, 2010) and Mycobacterium bovis BCG (Kyei et al, 2006) and aids their survival in macrophages but its role in phagosomes containing fungal pathogens is not known. Here, an important role for Rab14 in protecting macrophages against hyphal mediated lysis by C. albicans is demonstrated. Macrophages were transfected to express eGFP-Rab14, or dominant negative variants (eGFP-Rab14 S25N and eGFP-Rab14N124I); or were transfected with anti-Rab14 siRNA; then infected with live C. albicans and observed using sophisticated live cell imaging and analysis tools. Phagosomes containing live C. albicans became transiently Rab14-positive within 2 minutes following engulfment. Interestingly, a prolonged retention of Rab14 on phagosomes depended on C. albicans morphology. Phagosomes containing hyphal forms retained Rab14 for twice as long as for phagosomes containing the yeast form. Depletion of endogenous Rab14 did not affect macrophage migration towards C. albicans, the rate of engulfment or acquisition of markers of early phagosome maturation to phagosomes containing C. albicans. Furthermore, reduced Rab14 expression did not influence the kinetics of Rab5 localisation to phagosomes in macrophages that were co-transfected. Importantly, partially depleting Rab14 delayed the appearance of late phagosome maturation indicators LAMP1 and activated cathepsin in phagosomes containing live C. albicans.Rab14 knockdown was associated with a significant increase in macrophage killing by C. alibica The data presented in this thesis demonstrate the dynamic relationship between host and pathogen which can be visualised in real time at the level of individual phagosomes. Rab14 plays an important role during phagosome maturation which impacts on the later stages of phagosome maturation and is important for phagocyte survival after phagocytosis of C. albicans.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:629398 |
| Date | January 2014 |
| Creators | Okai, Blessing |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=214847 |
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