title: Modulation of cellular phosphatidylinositol 3-phosphate levels in primary macrophages affects heat-killed but not viable Mycobacterium avium's transport through the phagosome maturation process.
abstract: Most disease causing mycobacteria are intramacrophage pathogens which replicate within nonacidified phagosomes that can interact with the early endosomal network but fail to mature to a phagolysosome. The mycobacterial phagosome retain some proteins required for fusion with endocytic vesicles including Rab5 but lack others such as early endosomal autoantigen 1 (EEA1). As the membrane lipid phosphatidylinositol 3-phosphate (PtdIns-3-P) is required for EEA1 membrane association and phagosome maturation, it may be a potential target of pathogenic mycobacteria. To test this hypothesis, macrophage cellular levels of PtdIns-3-P were altered by retroviral introduction of the type III Phosphoinositide 3-Kinase (VPS34) and the PtdIns-3-P phosphatase myotubularin 1 (MTM1). By utilizing the PtdIns-3-P-specific probes FYVE and PX coupled to EGFP (EGFP-2-FYVE and EGFP-PX, respectively), the expression of PtdIns-3-P on the mycobacterial phagosome was addressed. All phagosomes containing viable Mycobacterium avium stained positive for EGFP-2-FYVE and EGFP-PX despite obvious differences in PtdIns-3-P concentrations in cells expressing MTM1 or VPS34. Altering PtdIns-3-P cellular concentrations did not affect trafficking of live bacilli. However, a significant increase in the transport of killed bacilli to a late endosomal/lysosomal compartment was observed in VPS34-compared to MTM1-transduced macrophages. Therefore, although overexpression of PdtIns-3-P in macrophages can facilitate phagosome maturation, its effect on phagosomes containing viable M. avium was negligible.
authors.affiliation: Department of Biological Sciences, Center for Tropical Disease Research and Training, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Kelley
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Schorey
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D015415:Biomarkers; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D006358:Hot Temperature; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009162:Mycobacterium avium / Q000166:cytology / Q000502:physiology*; D010588:Phagosomes / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015415:Biomarkers; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011993:Recombinant Fusion Proteins; C055525:phosphatidylinositol 3-phosphate; C099588:myotubularin