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 fusion of lysosomes containing bactericidal factors with phagosomes, and several intracellular pathogens are able to inhibit this fusion event. In this study, we report the reconstitution of phagosome-lysosome fusion in vitro, using an assay based on resonance energy transfer between fluorescent phospholipid analogues that were inserted into whole human NB4-neutrophil. membranes from liposomes containing positively charged lipids. Cytosol was required for fusion, and fusion was stimulated 3-fold if this cytosol had been prepared from neutrophils activated by using opsonized zymosan or a combination of the calcium ionophore (A23187) and phorbol myristate acetate (PMA). Fusion was inhibited by the addition of PPI, an inhibitor of Src family protein kinases, or GTP gammaS. We have previously reported that the biogenesis of phagolysosomes in human neutrophils is inhibited by mycobacteria. Here we show that cytosol from cells having internalized live (not heat-killed) Mycobacterium smegmatis or cytosol simply incubated with mycobacteria inhibited fusion, indicating that soluble factors are involved in mycobacterial inhibition of phagosome-lysosome fusion.