Host cell death in Legionella pneumophila pathogenesis and immunity
Legionella pneumophila is an intracellular pathogen that causes a severe, atypical pneumonia termed Legionnaires' disease. Upon entering the host cell, L. pneumophila resides in a membrane-bound vacuole, in which the bacterium evades lysosomal fusion and replicates. The establishment of the vacuole requires the Dot/Icm (Defect in organelle trafficking/ intracellular multiplication) transport system, which translocates a large number of substrates into host cells to re-orchestrate various cellular processes, such as intracellular trafficking, protein synthesis and host cell death pathways. Therefore, a key step in understanding the biology of Legionella is to dissect the mechanisms of action of the Dot/Icm substrates. By fusing the gene of interest with β-lactamase, we determined the transfer of the fusions into mammalian cells using the β-lactamase reporter substrate CCF4-AM. 164 Dot/Icm substrates were identified in this screening, which led to the expansion of the list of known Dot/Icm substrates by 70 effectors. These efforts have facilitated future studies on the biology of L. pneumophila and its interaction with hosts. Taking advantage of the expanded list of L. pneumophila Dot/Icm effectors, we initiated a screening to survey all known effectors for the ability to activate caspase-3. Our screening led to the discovery that at least five Dot/Icm substrates, Lpg0716, Lpg0898, Lpg1831, Lpg1625 and Lpg2716, activate caspase-3 when ectopically expressed in mammalian cells. We further demonstrated that one of these effectors, VipD (lpg2831), is a phospholipase A2 that hydrolyzes the phosphatidylethanolamine (PE) and phosphatidylcholine (PC) on the mitochondrial membrane in a manner that appears to require host cofactor(s). The lipase activity is essential to its ability to activate caspase-3 via the production of free fatty acids and 2-lysophospholipids, which destabilize the mitochondrial membranes. Mitochondria membrane destabilization may contribute to cytochrome c release and subsequent activation of caspase-3. To investigate how primary macrophages respond to Legionella environmental isolates, which are responsible for most of the Legionnaires' disease outbreaks so far, we analyzed the intracellular growth of a few such isolates in A/J mouse macrophages, which is permissive to most, if not all, laboratory strains. None of the Legionella environmental isolates were able to replicate in these macrophages. Further investigation into one of the isolates, LPE509, revealed that such growth restriction is not due to the lack of important pathogenic determinants, as this strain is competent of replication in two protozoan hosts and the human macrophage U937 cell. Moreover, the inability of LPE509 to replicate intracellularly is accompanied by host cell death of unique features, as it is independent of most cell death signaling components known to control Legionella infection. These results together with the conservation of rpsL gene across diverse bacterial species prompted us to test whether the RpsL protein per se is responsible for the induction of host cell death. Indeed, delivery of RpsL into mouse macrophages elicits a noncanonical form of cell death involved in the permeabilization of lysosomal membranes. Upon the loss of lysosome membrane integrity, various hydrolases are released into the cytosol, where they process diverse substrates and cause cell death. Amongst these hydrolases, cathepsin B plays an important role in RpsL induced cell death, as genetic ablation of this gene dampens host cell death induced by recombinant RpsLWT or Legionella expressing wild type RpsL. Taken together, our results suggested that RpsL is a microbial associated molecular pattern (MAMP) that triggers a unique cell death pathway in mouse macrophages, which limits the spread of invading bacterial pathogens. (Abstract shortened by UMI.)
Zhou, Purdue University.
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