title: Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome.
abstract: The Dot/Icm type IV secretion system of Legionella pneumophila is essential for evasion of endocytic fusion and for activation of caspase-3 during early stages of infection of macrophages, but the mechanisms of manipulating these host cell processes are not known. Here, we show that caspase-3 activation by L. pneumophila is independent of all the known apoptotic pathways that converge on the activation of caspase-3. The cytoplasmic proteins IcmS, IcmR and IcmQ, which are involved in secretion of Dot/Icm effectors, are required for caspase-3 activation. Pretreatment of U937 macrophages and human peripheral blood monocytes (hPBM) with the capase-3 inhibitor (DEVD-fmk) or the paninhibitor of caspases (Z-VAD-fmk) before infection blocks intracellular replication of L. pneumophila in a dose-dependent manner. Inhibition of caspase-3 results in co-localization of the L. pneumophila-containing phagosome (LCP) with the late endosomal/lysosomal marker Lamp-2, and the LCP contains lysosomal enzymes, similar to the dotA mutant, which is defective in caspase-3 activation. However, activation of caspase-3 before infection does not rescue the replication defect of the dotA mutant. Interestingly, inhibition of caspase-3 after a 15 or 30 min infection period by the parental strain has no detectable effect on the formation of a replicative niche. The Dot/Icm-mediated activation of caspase-3 by L. pneumophila specifically cleaves, in a dose- and time-dependent manner, the Rab5 effector Rabaptin-5, which maintains Rab5-GTP on the endosomal membrane. In addition, PI3 kinase, which is a crucial effector of Rab5 downstream of Rababptin-5, is not required for intracellular replication. Using single-cell analysis, we show that apoptosis is not evident in the infected cell until bacterial replication results in > 20 bacteria per cell. We conclude that activation of caspase-3 by the Dot/Icm virulence system of L. pneumophila is essential for halting biogenesis of the LCP through the endosomal/lysosomal pathway, and that this is associated with the cleavage of Rabpatin-5.
authors.affiliation: Department of Microbiology and Immunology, University of Louisville College of Medicine, Louisville, KY 40292, USA.
authors.forename: Maëlle
authors.initials: M
authors.lastname: Molmeret
authors.forename: Steven D
authors.initials: SD
authors.lastname: Zink
authors.forename: Lihui
authors.initials: L
authors.lastname: Han
authors.forename: Alaeddin
authors.initials: A
authors.lastname: Abu-Zant
authors.forename: Rexford
authors.initials: R
authors.lastname: Asari
authors.forename: Dina M
authors.initials: DM
authors.lastname: Bitar
authors.forename: Yousef
authors.initials: Y
authors.lastname: Abu Kwaik
mesh_terms: D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D017209:Apoptosis; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D018929:Cell Culture Techniques; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D016952:Legionella pneumophila / Q000472:pathogenicity*; D051907:Lysosomal Membrane Proteins; D008264:Macrophages / Q000201:enzymology / Q000382:microbiology*; D018832:Molecular Chaperones / Q000235:genetics / Q000502:physiology; D009000:Monocytes / Q000201:enzymology / Q000382:microbiology*; D010588:Phagosomes / Q000378:metabolism / Q000382:microbiology*; D020298:U937 Cells; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D014774:Virulence; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D001426:Bacterial Proteins; D015853:Cysteine Proteinase Inhibitors; C432955:ICMQ protein, Legionella pneumophila; C432954:IcmR protein, Legionella pneumophila; D051907:Lysosomal Membrane Proteins; D018832:Molecular Chaperones; C097227:RABEP1 protein, human; D033921:Vesicular Transport Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020691:rab GTP-Binding Proteins