Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization

Abstract

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

Figure 1. Caspase-11 is dispensable for caspase-1 activation and interacts with members of the Nlrc4 inflammasome

(a) Wild-type (WT), caspase-1-deficient (Casp1^−/−) and Caspase-11-deficient (Casp4^−/−) BMDMs were infected with L. pneumophila (Leg), its corresponding flagellin mutant (Fla) for 2 hrs, or left untreated (NT). Cell lysates were immunoblotted for pro-, cleaved (caspase-1) Casp1, (caspase-11) Casp11 and actin. (b) WT, Nlrc4^−/−, Pycard^−/− (Asc-deficient) and A/J (express mutant Naip5) BMDMs were uninfected (NT) or infected with Leg and the expression of caspase-11 was examined by Western blot. (c) WT and Casp4^−/− BMDMs were infected with Listeria monocytogenes (Lm), Francisella novicida (Fn), and Salmonella Typhimurium (St) for 2 hrs or left untreated (NT). (a, b, and c) Cell lysates were immunoblotted for pro-, and cleaved Casp1, and Casp11 and actin. (d) WT and Casp4^−/− BMDMs were untreated (NT) or infected with Leg for 4 hrs. (e) WT macrophages were untreated (NT) or infected with Leg, Fla for 4 hrs. (d and e) Casp11 was then immunoprecipitated from cell lysates. The Western blots of cell lysates and of immune-complexes (IP) were probed with Nlrc4, Casp1, Asc and Casp11 antibodies. Blots are representative of three independent experiments.

Figure 2. Caspase-11-deficient (Casp4^−/−) macrophages allow L. pneumophila intracellular replication

(a) Wild-type (WT), caspase-11-deficient (Casp4^−/−) and caspase-1-deficient (Casp1^−/−) murine BMDMs were infected with L. pneumophila (Leg) and colony forming units (CFUs) were enumerated at 1, 24, 48 and 72 hrs. Data are representative of three independent experiments and presented as means ± S.D. Asterisks indicate significant differences from WT macrophages (***P<0.001). (b) Confocal microscopy of Leg-infected WT or Casp4^−/− BMDMs after 24 hrs. Nuclei are stained blue with DAPI and Leg express green florescent protein (GFP). White arrows indicate the sites of Leg. (c and d) WT, Casp1^−/−, and Casp4^−/− BMDMs were nucleofected with plasmid harboring Casp4 (PL- Casp11) or empty vector (PL) for 24 hrs. (c) BMDMs were infected with L. pneumophila and CFUs were enumerated at 1, 24, 48 and 72 hrs. (d) Samples were lysed and immunoblotted for caspase-11 expression.

Figure 3. Caspase-11 is activated during L. pneumophila infection and restricts infection in vivo

(a) Wild-type (WT) BMDMs were infected with L. pneumophila (Leg) or its isogenic flagellin mutant (Fla), lysed (cell lysates), then mixed with biotinylated-YVAD-CMK, immunoprecipitated (IP), processed for Western blot with caspase-11 antibodies. (b) WT macrophages were nucleofected with siRNA specific to Casp4 (siRNA-Casp4) or siRNA control (siRNA-CT) then lysed and processed for Western blots to detect the expression of caspase-11 (Casp11) protein. (c) Macrophages were treated as in (b) then, infected with L. pneumophila and colony forming units (CFUs) were enumerated at 1, 24, 48 and 72 hrs. Data are representative of three independent experiments ± S.D. (d and e) WT and caspase-11-deficient (Casp4^−/−) mice were infected intratracheally with L. pneumophila then CFUs recovered from homogenized lungs were enumerated and expressed as CFU per gram of lung tissue at 4 hrs (d) and 48 hrs (e). Data are represented as the means of data obtained from 4 mice ±S.D. Asterisks indicate significant differences (**P<0.01; ***P<0.001).

Figure 4. Caspase-11 promotes L. pneumophila degradation in macrophages

(a) Wild-type (WT) and caspase-1-deficient (Casp1^−/−) BMDMs were nucleofected with plasmid carrying Casp4 gene (PL- Casp11) or vector alone (PL), and infected with L. pneumophila and colony forming units (CFUs) were enumerated at 1, 24, 48, and 72 hrs. (b) WT and caspase-11-deficient (Casp4^−/−) macrophages were infected with L. pneumophila for 4 hrs and processed for electron microscopy. Black arrow indicates internalized L. pneumophila showing irregular contour, and black arrowhead indicates intact L. pneumophila (magnification 80,000). (c) The percent of degraded bacteria in WT and Casp4^−/− BMDMs were quantified by confocal microscopy using specific L. pneumophila antibody. (d) WT and Casp4^−/− macrophages were infected with the SSK strain of L. pneumophila that responds to IPTG by expressing GFP. The percentage of L. pneumophila not responding to IPTG (GFP^−/−) was quantified by confocal microscopy. Data in a, c and d are representative of three independent experiments ±S.D. Asterisks indicate significant differences (*P< 0.05; **P<0.01; ***P<0.001).

Figure 5. Caspase-11 activity is required to promote the fusion of the lysosome with phagosomes harboring L. pneumophila but not those harboring E. coli

(a) WT and caspase-11-deficient (Casp4^−/−) BMDMs were infected with L. pneumophila (Leg) constitutively expressing GFP. Fixed samples were processed for confocal microscopy. (b and e) The colocalization of the bacteria with lysotracker red was enumerated. The sites of colocalization are indicated with white arrows (a and f). (c) Cells treated as in (a) were fixed and colocalization of Leg with the endocytic marker LAMP-1 was quantified. (d) WT and Casp4^−/− BMDMs were nucleofected with vector alone (PL), plasmid carrying native Casp4 gene (PL- Casp11), or plasmid carrying mutant Casp4 gene (PL-inactive Casp11), and infected with Leg. (e and f) WT and Casp4^−/− BMDMs were infected with GFP constitutively expressing Escherichia coli (E. coli). Data are representative of three independent experiments and presented as the means ± S.D. Asterisks indicate significant differences (*P<0.05; **P<0.01; ***P<0.001).

Figure 6. Caspase-11 is required for the dynamic formation of polymerized actin around phagosomes

(a–c) WT, caspase-11-deficient (Casp4^−/−) (a and b), and Casp1^−/− BMDMs (c) were infected with L. pneumophila (Leg) constitutively expressing GFP. Polymerized actin was stained with rhodamine-phalloidin. (a) The amount of rhodamine-phalloidin within equal areas was quantified by confocal microscopy and expressed as arbitrary units. (b) The percentage of phalloidin-labelled Leg-containing phagosomes was quantified by confocal microscopy. (c) Confocal microscopy showing rhodamine-phalloidin staining (red) around GFP-expressing (green) Leg. Phagosomes containing degraded bacteria are heavily labeled for polymerized actin (white arrow heads). Data in a and b are representative of three independent experiments and presented as the means ±S.D. Asterisks indicate significant differences (*P<0.05; **P<0.01; ***P<0.001).

Figure 7. Caspase-11 is required for the phosphorylation of cofilin and interacts with actin upon L. pneumophila infection

(a) WT and caspase-11-deficient (Casp4^−/−) BMDMs were infected with L. pneumophila (Leg) for 2 and 4 hrs or left untreated (NT). BMDMs lysates were immunoblotted with antibodies against phosphorylated cofilin (P-cofilin), cofilin and actin. (b) WT and Casp4^−/− BMDMs were infected with Leg or left NT, lysed (cell lysates), and immunoprecipitated (IP) with beads coated with caspase-11 antibody and immunoblotted with actin antibody.