Expression and Localisation of the Mycobacterium marinum Potential Virulence Factor, MPM70 during Infection: Evidence for a Post-Phagosomal Role in Pathogenesis
thesisposted on 03.02.2012, 14:07 by Alistair Frank Ray
During infection, MPT70 is one of the most highly upregulated Mycobacterium tuberculosis genes, implying an important role in virulence. To track expression of MPT70 during macrophage infection, a Mycobacterium marinum: J774A.1 macrophage infection model was used with a custom polyclonal antibody raised against purified full-length, folded MPM70 without its N-terminal signal sequence (88% homology to M. tuberculosis MPT70). Immunofluorescence studies showed that MPM70 is localised in large, non-uniformly arranged buttresses along the mycobacterial cell surface. Analysis of representative confocal microscopy images revealed that MPM70 expression was lowest at initial infection levels of 1-5 mycobacteria per host cell, but was greatly upregulated at mycobacterial densities of over 6, where expression remained constant in around 20% of the infected cells up to mycobacterial densities of greater than 35. This implies that MPM70 expression is independent of mycobacterial density following the initial stages of infection and may be triggered by individual mycobacteria contacting a specific cytoplasmic host cell factor. Cytoplasmic tracks of MPM70 were a rare observation, but suggest that the protein may act as a molecular intermediate between the mycobacterial surface and a cytoplasmic host cell protein. Structural analyses and comparisons to eukaryotic homologues such as fasciclin and βig-H3 reveal two potential interaction faces on opposite sides of MPT70, which supports the hypothesis that MPT70/MPM70 may act as a ‘molecular bridge’ between two proteins. Evidence of MPM70 production in cell infections using a ΔRD1 strain of M. marinum showed that MPM70 expression is RD1 independent. We used the lysosomal marker LAMP-1 and the lypophilic dye DiIC18(5)-DS to show that expression of MPM70 occurs after mycobacteria have escaped from the phagolysosome. A possible role for MPM70 in the modulation of host cell actin was investigated, but fluorescent phalloidin staining showed no co-localisation with expressed MPM70.