Abstract
The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase-1−/− and Nlrc4−/− allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc−/− macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.
Highlights
The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc encoded by the Pycard gene) is an adaptor molecule that mediates inflammatory and apoptotic signals and is predominantly expressed in monocytes and mucosal epithelial cells (Taniguchi and Sagara, 2007; Hasegawa et al, 2009)
After depletion of Asc, cells were infected with L. pneumophila and the bacterial replication was assessed by counting colony-forming units (CFU)
In WT mouse macrophages, depletion of Asc did not have an effect on the bacterial growth compared to that of cells treated with control siRNA or untreated cells (Figure 1D)
Summary
The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc encoded by the Pycard gene) is an adaptor molecule that mediates inflammatory and apoptotic signals and is predominantly expressed in monocytes and mucosal epithelial cells (Taniguchi and Sagara, 2007; Hasegawa et al, 2009). Asc contains an N-terminal pyrin/PAAD (PYD) death domain and a C-terminal CARD protein–protein interaction domain (CARD; Masumoto et al, 1999, 2001; Liepinsh et al, 2003; Stehlik et al, 2003) Both domains enable Asc to recruit other PYD and CARD-containing proteins through homotypic protein–protein interactions (Fernandes-Alnemri et al, 2007; Mariathasan, 2007). Asc is able to link caspase-1 to NOD-like receptors (NLRs) via its CARD domain, leading to the activation of caspase-1 (Srinivasula et al, 2002; Fernandes-Alnemri et al, 2007; Fernandes-Alnemri and Alnemri, 2008). Many Gram-negative bacteria, such as Salmonella typhimurium, Pseudomonas aeruginosa, Shigella flexneri, and Legionella pneumophila, are recognized in murine macrophages by the NLR Nlrc4/Ipaf leading to caspase-1 activation through the inflammasome (Abdelaziz et al, 2010; Amer, 2010)
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