Fusion of Human Neutrophil Phagosomes with Lysosomes in Vitro: INVOLVEMENT OF TYROSINE KINASES OF THE Src FAMILY AND INHIBITION BY MYCOBACTERIA

Pascale Peyron,Isabelle Maridonneau-Parini,Toon Stegmann

doi:10.1074/jbc.m104399200

Abstract

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 PP1, an inhibitor of Src family protein kinases, or GTPgammaS. 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.

Highlights

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
We have previously reported that the biogenesis of phagolysosomes in human neutrophils is inhibited by mycobacteria
Cells labeled using a pulse-chase protocol were activated by incubation for 10 min at 37 °C in the presence of A23187 and phorbol myristate acetate (PMA) according to Welch and Maridonneau-Parini [14], a treatment that activates NADPH oxidase and stimulates lysosome exocytosis

Summary

EXPERIMENTAL PROCEDURES

Cell Labeling and Organelle Purification—Dry lipid films were produced from a mixture of N-NBD-PE, N-Rhodamine-PE, and 1,2-dioleoyl-3-N, N,N,-trimethylammoniumpropane (DOTAP) (2:2:1 molar ratio; Avanti Polar Lipids, Birmingham, AL) and taken up into doubledistilled water to produce liposomes. 156 nmol of the lipids in 500 ␮l of water were added to 2.5 ϫ 107 cells in a volume of 25 ml of tissue culture medium (MEM), buffered with 10 mM HEPES pH 7.4 (MEM/HEPES) for 15 min at 37 °C. The cells were washed by centrifugation and incubated in culture medium for 50 min at 37 °C, after which A23187 and PMA were added to final concentrations of 5 ␮M and 100 ng/ml, respectively. After an additional 10 min at 37 °C, the cells were collected by centrifugation, suspended in 1 ml of buffer and subjected to nitrogen at 375 psi for 10 min [14].

The abbreviations used are

RESULTS

DISCUSSION