Fig. 7.

ΔfbpA phagosomes acquire late endosomal maturation markers but exclude lysosomal markers. MΦs were infected with gfpΔfbpA, gfpH37Rv and gfpBCG strains for 4 h, washed and incubated for 24 h. MΦs were fixed, permeabilized and stained for three markers of late endosomes (lysosome-associated membrane protein 1, LAMP-1; src family kinase, Hck) and lysosomes (CD63 and rab7) using specific antibodies and counterstained with anti-Ig-Texas red conjugates. Phagosomes were examined for colocalization (arrow and arrowhead).

A. LSM profile illustrates that gfpΔfbpA has an enhanced colocalization (arrow) with the Hck.

B. Illustration that gfpΔfbpA largely excludes rab7.

C. Per cent phagosomes positive for fluorescence colocalization with the markers was determined by using LSM. gfpΔfbpA phagosomes are more enriched for LAMP-1 and Hck but not CD63 and rab7. gfpBCG and gfpH 37Rv also acquire LAMP-1 to a lower level but exclude Hck, CD63 and rab7. Scoring from three separate experiments was averaged and significance determined using Student's t-test.

D. MΦs were infected with mycobacteria at an moi of 1:1 and phagosomes were purified on sucrose gradients at 24 h post infection. Latex beads (LB) were internalized for 4 h and phagosomes prepared as a positive control. Phagosomal proteins were then analysed using Western blot with specific antibodies followed by anti-Ig conjugates and HRP-dependent chemiluminescence. LB phagosomes acquire nearly all docking proteins along with CD63 and rab7, the specific marker of lysosomes. gfpΔfbpA phagosomes acquire multiple markers that suggest an ability to fuse with early and recycling endosomes such as rab5A, rab5B and rab11. While they acquire late endosomal markers such as Hck, they exclude lysosome-specific markers like CD63 and rab7. LAMP-1 and phosphatidyl inositol-3-kinase are also enriched on LB and gfpΔfbpA phagosomes compared with gfpBCG and gfpH 37Rv.