Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome

Cell Microbiol. 2004 Jan;6(1):33-48. doi: 10.1046/j.1462-5822.2003.00335.x.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, CD / immunology
  • Antigens, CD / metabolism
  • Apoptosis
  • Bacterial Proteins / genetics
  • Bacterial Proteins / physiology
  • Caspase 3
  • Caspases / metabolism*
  • Cell Culture Techniques
  • Cysteine Proteinase Inhibitors / pharmacology
  • Enzyme Activation
  • Humans
  • Legionella pneumophila / pathogenicity*
  • Lysosome-Associated Membrane Glycoproteins
  • Macrophages / enzymology
  • Macrophages / microbiology*
  • Molecular Chaperones / genetics
  • Molecular Chaperones / physiology
  • Monocytes / enzymology
  • Monocytes / microbiology*
  • Phagosomes / metabolism
  • Phagosomes / microbiology*
  • U937 Cells
  • Vesicular Transport Proteins / metabolism*
  • Virulence
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Antigens, CD
  • Bacterial Proteins
  • Cysteine Proteinase Inhibitors
  • ICMQ protein, Legionella pneumophila
  • IcmR protein, Legionella pneumophila
  • Lysosome-Associated Membrane Glycoproteins
  • Molecular Chaperones
  • RABEP1 protein, human
  • Vesicular Transport Proteins
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • rab GTP-Binding Proteins