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Title: Mycobacterial resuscitation promoting factors : roles and mechanisms in infected macrophages
Author: Iakobachvili, Nino
ISNI:       0000 0004 5351 219X
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2015
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Members of the Mycobacterium tuberculosis complex are the causative agents of tuberculosis, a major global health threat to human populations. The majority of infected individuals harbour a latent infection where bacteria persist in a non-replicating, dormant state. Resuscitation promoting factors (Rpfs) are secreted proteins whose peptidoglycan hydrolyzing abilities have been correlated with the reactivation of dormant bacteria; however, the precise molecular mechanisms underlying this remain poorly understood. The individual roles, localisation and expression patterns of Mycobacterium marinum Rpf homologues during macrophage infection were investigated by immunofluorescence using custom Rpf-specific polyclonal antibodies and confocal microscopy. We found Rpfs to associate to the polar ends of bacteria, along the lengths of the bacterial surface and free in the cytoplasm of infected macrophages suggesting a role for these proteins in apical extension and peptidoglycan biosynthesis. No distinct patterns of Rpf localisation were observed in relation to phagosomal mycobacteria. Microscopy based semi-quantification of Rpf expression levels during macrophage infection, and analysis of in vitro grown bacteria from logarithmic phase by flow cytometry identified that only a subset of mycobacteria produce Rpf at detectable levels. Significant protein sequence homology between RpfA and Listeria monocytogenes ActA suggested that RpfA could play a similar role in actin polymerisation and bacterial motility in the macrophage cytoplasm as ActA. However, under the conditions tested, ΔrpfA M. marinum did not show any differences to wildtype during infection of murine macrophages. Recombinant ActA was demonstrated to have muralytic activity, a previously uncharacterised property of this protein suggesting a function beyond the polymerisation of host cell actin.
Supervisor: Mukamolova, Galina; Carr, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available