Fusion of human neutrophil phagosomes with lysosomes in vitro - Involvement of tyrosine kinases of the Src family and inhibition by mycobacteria

The intracellular killing of microorganisms in phagocytes involves the fusi on of lysosomes containing bactericidal factors with phagosomes, and severa l intracellular pathogens are able to inhibit this fusion event. In this st udy, we report the reconstitution of phagosome-lysosome fusion in vitro, us ing an assay based on resonance energy transfer between fluorescent phospho lipid analogues that were inserted into whole human NB4-neutrophil. membran es from liposomes containing positively charged lipids. Cytosol was require d for fusion, and fusion was stimulated 3-fold if this cytosol had been pre pared from neutrophils activated by using opsonized zymosan or a combinatio n of the calcium ionophore (A23187) and phorbol myristate acetate (PMA). Fu sion was inhibited by the addition of PPI, an inhibitor of Src family prote in kinases, or GTP gammaS. We have previously reported that the biogenesis of phagolysosomes in human neutrophils is inhibited by mycobacteria. Here w e show that cytosol from cells having internalized live (not heat-killed) M ycobacterium smegmatis or cytosol simply incubated with mycobacteria inhibi ted fusion, indicating that soluble factors are involved in mycobacterial i nhibition of phagosome-lysosome fusion.