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Activation of caspase‐3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella‐containing phagosome
7 auth. M. Molmeret, S. Zink, Lihui Han, A. Abu‐Zant, R. Asari, D. M. Bitar, ...
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…
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.
Published in
Cellular Microbiology
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3
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6 | 2004 |
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