title: Lack of fusion of azurophil granules with phagosomes during phagocytosis of Mycobacterium smegmatis by human neutrophils is not actively controlled by the bacterium.
abstract: Biogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as beta-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellular Mycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of beta-glucuronidase, indicating that M. smegmatis does not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellular M. smegmatis opsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose that M. smegmatis does not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique and Université Paul Sabatier, UMR 5089, 31077 Toulouse, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Cougoule
authors.forename: Patricia
authors.initials: P
authors.lastname: Constant
authors.forename: Gilles
authors.initials: G
authors.lastname: Etienne
authors.forename: Mamadou
authors.initials: M
authors.lastname: Daffé
authors.forename: Isabelle
authors.initials: I
authors.lastname: Maridonneau-Parini
mesh_terms: D000199:Actins / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000276:immunology*; D008561:Membrane Fusion; D020102:Mycobacterium smegmatis / Q000276:immunology*; D009504:Neutrophils / Q000276:immunology*; D010587:Phagocytosis / Q000276:immunology*; D010588:Phagosomes / Q000276:immunology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051572:Proto-Oncogene Proteins c-hck
chemical_list: D000199:Actins; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; D005966:Glucuronidase