title: Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles.
abstract: Listeria monocytogenes (Lm) evade microbicidal defences inside macrophages by secreting a pore-forming cytolysin listeriolysin O (LLO), which allows Lm to escape vacuoles. LLO also inhibits Lm vacuole fusion with lysosomes, which indicates LLO alters vacuole chemistry prior to release of Lm into cytoplasm. Using fluorescent probes to measure membrane permeability, calcium and pH, we identified small membrane perforations in vacuoles containing wild-type but not LLO-deficient (hly-) Lm. The small membrane perforations released small fluorescent molecules and persisted for several minutes before expanding to allow exchange of larger fluorescent molecules. Macropinosomes and hly- Lm vacuoles acidified and increased their calcium content ([Ca2+]vac) within minutes of formation; however, the small perforations made by LLO-expressing bacteria increased vacuolar pH and decreased [Ca2+]vac shortly after infection. Experimental increases in vacuolar pH inhibited Lm vacuole fusion with lysosomes. The timing of perforation indicated that LLO-dependent delays of Lm vacuole maturation result from disruption of ion gradients across vacuolar membranes.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA.
authors.forename: Lee M
authors.initials: LM
authors.lastname: Shaughnessy
authors.forename: Adam D
authors.initials: AD
authors.lastname: Hoppe
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Christensen
authors.forename: Joel A
authors.initials: JA
authors.lastname: Swanson
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000382:microbiology* / Q000502:physiology; D008089:Listeria monocytogenes / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000502:physiology; D008264:Macrophages / Q000382:microbiology / Q000502:physiology; D008561:Membrane Fusion; D051379:Mice; D010539:Permeability; D014617:Vacuoles / Q000502:physiology*
chemical_list: D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes; D002118:Calcium