legionella infection  (ATCC)

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Wild-type (WT) and Casp-1 −/− Casp-11 Tg macrophages were infected with ( a ) GFP-expressing Legionella ( L.p .) or ( c ) Escherichia ( E. ) coli . Macrophages were stained with LysoTracker® red that accumulates in acidic compartments. Quantification of colocalization of LysoTracker® with ( b ) GFP- L.p. and ( d ) GFP- E.coli . Mean percent colocalization ± SEM from three biological replicates are represented. ( b ) significance, indicated by (*), was obtained by performing Student’s unpaired t -test. p = 0.0002 is ***, p = 0.01 is **, and p = 0.0113 is *.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Infection, Expressing, Staining

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Colony forming unit (CFU) assay of Legionella replication in WT and Casp-1 −/− Casp-11 Tg ( a ) in vitro after 1, 24, 48, 72 hrs post infection and ( b ) in vivo from lungs of WT and Casp-1 −/− Casp-11 Tg mice at 4 and 48 hrs after infection. ( c ) Live/Dead® immunofluorescence assay on WT, Casp-11 −/− , Casp-1 −/− Casp-11 Tg BMDMs infected (2 hrs) or not (NT) with Legionella . ( d ) Quantification of Live/Dead® stain after 1, 2, and 4 hrs after infection and mean percent dead macrophages ± SEM from three biological replicates are represented. ( a ) significance, indicated by (*), was obtained by performing Student’s unpaired t -test. 24 hrs p = 0.000098 is ****, 48 hr p = 0.000087 is ****, 72 hr p = 0.0017 is **, respectively. ( b ) 48 hrs p = 0.021 is *. ( d ) significance of the Live/Dead assay was calculated by one-way ANOVA with Student’s t -test post-test. *** is p < 0.001 and ** is p < 0.01.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Colony-forming Unit Assay, In Vitro, Infection, In Vivo, Immunofluorescence, Staining, Live Dead Assay

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Confocal analysis of WT, Casp-11 −/− , and Casp-1 −/− Casp-11 Tg macrophages double-labeled with Alexa Fluor 568 phalloidin (stains F-actin) and Alexa Fluor 488 DNAse I (stains G-actin) after 2 hrs infection with ( a ) Legionella ( L.p. ) or ( b ) Escherichia (E. ) coli . F-actin structures in the vicinity of the Legionella vacuole in WT macrophages are indicated by white arrow, white tabs indicate their absence. Data are representative of three biological replicates performed independently.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Labeling, Infection

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Immunoblot analysis of phospho-cofilin (P-cofilin) after infection with Legionella in ( a ) WT, Casp-11 −/− , and Casp -1 −/− Casp -11 Tg . ( b ) Nlrc4 −/− and Naip5 −/− macrophages. ( c ) P-cofilin immunoblots of WT cells infected with parental Legionella ( L.p .), the T4SS ( dotA − ) mutant and flagellin ( flaA − ) mutant for 1, 2, and 4 hrs. ( d ) Lysates from WT macrophages infected with live and heat-killed Legionella analyzed for P-cofilin at 1 and 2 hrs after infection. Blots are representative of at least three independent experiments.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Western Blot, Infection, Mutagenesis

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Rac GTPase activity ( a ) and RhoA GTPase activity ( b ) from WT and Casp-11 −/− macrophage lysates after infection with Legionella using specific G-LISA activation assay (Cytoskeleton) 0.25, 0.5, 1, and 2 hrs post infection. All data are representative of at least three independent experiments. G-LISA assays analyzed for significance by Student’s unpaired t -test. *** is p < 0.001, ** is p < 0.01, and * is p<0.05.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Activity Assay, Infection, Activation Assay

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: WT and Casp-1 −/− Casp-11 Tg macrophages assayed for activity of ( a ) Rac/Cdc42 by Western blot analysis using phospho-Rac/Cdc42 antibodies 1 and 2 hrs post infection, ( b ) RhoA GTPase using specific G-LISA activation 0.25, 0.5, 1, and 2 hrs after infection, and ( c ) Slingshot by immunoblot using phospho-Slingshot specific antibody after 1 and 2 hrs infection with Legionella . Data are representative of at least three independent experiments. ( b ) Student’s unpaired t -test was performed to determine significance, time points exhibited no significance (NS).

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Activity Assay, Western Blot, Infection, Activation Assay

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Immunoblot analysis of lysates of HEK293 cells transduced with lentiviral construct ( a ) containing enzymatically active and inactive (mut) caspase-11 red fluorescent plasmid after 1 h infection with Legionella with phospho-cofilin (P-cofilin) antibody ( b ). Blots are representative of three independent experiments.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Western Blot, Transduction, Construct, Plasmid Preparation, Infection

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Summary of caspase function during Legionella infection and outcomes.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Infection

Journal: Scientific Reports

Article Title: Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

doi: 10.1038/srep18479

Figure Lengend Snippet: Accordingly, caspase-11 promotes the phosphorylation of cofilin and actin depolymerization, whereas caspase-1 promotes the dephosphorylation of cofilin and actin polymerization. The dynamics of F/G actin ratio facilitates the fusion of the Legionella vacuole with lysosomes.

Article Snippet: Lysates from Legionella -infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations (RhoA, BK124 and Rac, BK128; Cytoskeleton).

Techniques: Phospho-proteomics, De-Phosphorylation Assay