| Legionella pneumophila (L. pneumophila ) is the causative agent of Legionellosis, two forms of respiratory illness: Pontiac Fever, an influenza-like self-limiting, febrile sickness that presents in healthy individual and Legionnaires' disease (LD), a potentially fatal pneumonia that affects immunocompromised individuals (HIV+, transplant, cancer, and COPD patients). Approximately, 18,000 people each year in the United States are hospitalized due to LD. Humans are prone to infection as the pathogen is ubiquitous in aquatic environments and has evolved a wide array of strategies to subvert and evade host cell immunity.;Clearance of L. pneumophila requires efficient phagolysosomal fusion. This allows for host cells to employ non-cell death-mediated defenses to destroy the intracellular organism by eliminating its replicative niche. Regulation of the host actin cytoskeleton is crucial for proper vesicle trafficking and fusion events. In this work, it was determined that activation of caspases-1 and -11 are required for trafficking and fusion during infection. The molecular mechanisms of caspase-mediated clearance are unknown. The objective of this study was to decipher the effects of caspase-1 and -11 on actin dynamics to promote phagolysosomal fusion and restriction of the bacteria.;Upon infection, Casp-1-/- and Casp-11-/- bone marrow-derived macrophages (BMDMs) were found to be permissive to L. pneumophila as these cells exhibited significantly lower amounts of colocalization of L. pneumophila -containing phagosomes and lysosomes compared to restrictive C57BL/6 (WT) cells. It was also determined that Casp-1-/- and Casp-11-/- macrophages exhibited diminished F/G-actin ratios compared to WT counterparts, while also failing to polymerize actin in the vicinity of L. pneumophila during infection. In elucidating the molecular mechanism, it was found that caspase-1 and -11 converge on an upstream regulator of actin dynamics: cofilin. During infection, caspase-1 promoted dephosphorylation (activation) of cofilin, while caspase-11 regulated its phosphorylation (inactivation). In addition, it was determined that upstream signaling molecules, RhoA and Slingshot, affected the activation of cofilin. The absence of caspase-11 resulted in significantly decreased RhoA GTPase activation, thereby keeping cofilin unphosphorylated. Also, caspase-1-deficient cells inhibited Slingshot phosphatase activity during infection, blocking cofilin dephosphorylation. Together, these molecules differentially modulated cofilin activation, affecting actin dynamics during L. pneumophila infection.;These data establish for the first time that inflammasome caspases differentially regulate actin polymerization during L. pneumophila infection by modulating F-actin assembly via the activation of cofilin. Understanding the novel molecular mechanisms of caspase-mediated regulation of the host cytoskeletal network will provide novel targets to develop therapeutic interventions for numerous infectious diseases. |
