Enteric pathogens deploy cell cycle inhibiting factors to block the bactericidal activity of Perforin-2
- Miller School of Medicine, University of Miami, United States
Figures
Figure 1
Domain organization of human C8a, C9, Perforin-1, and Perforin-2.
The pore forming components of complement (C8α and C9), Perforin-1, and Perforin-2 all contain membrane attack complex perforin (MACPF) domains and amino-terminal signal peptides (SPs). MACPF …
Figure 2
Perforin-2 co-immunoprecipitates with CRL subunits and is ubiquitylated.
RAW 264.7 cells were transfected with Perforin-2-GFP or GFP expression constructs and stimulated with IFN-γ prior to immunoprecipitation with anti-GFP. (A) Western blots probed with the indicated …
Figure 3
Perforin-2 activity is dependent upon its ubiquitylation by a CRL complex.
(A) siRNA efficiently and specifically knocks down expression of CUL1, UBC12, and Perforin-2 in IFN-γ stimulated CMT93 cells. (B) Western blots of immunoprecipitates from transfected CMT93 cells …
Figure 4
Conserved lysines within the carboxy-terminal domain of Perforin-2 are required for its ubiquitylation and bactericidal activity.
(A) Alignment of the predicted cytosolic domains of Perforin-2 (P2) molecules from select mammalian species. Dots denote identity to human Perforin-2. The three most conserved lysine residues are …
Figure 5 with 1 supplement
Cif blocks ubiquitylation of Perforin-2.
CMT93 cells were transfected with GFP or Perforin-2-GFP expression plasmids and stimulated with IFN-γ. As indicated, some transfected cells were also stimulated with LPS, infected with Yersinia …
Figure 5—figure supplement 1
CifYp expression.
Western blot demonstrating that both CifYp-FLAG and CifYp-C117A-FLAG are expressed in Y. pseudotuberculosis cif::aadA. Cloning vector pFLAG-CTC was included as a negative control.
Figure 6
The bacterial effector protein Cif blocks the bactericidal activity of Perforin-2.
Caco-2 cells, a human intestinal epithelial cell line, were transfected with Perforin-2 specific or scramble siRNAs. The cells were subsequently infected with EPEC strain E2348/69, which carries a …
Figure 7
Enzymatically inactive Cif cannot block Perforin-2-dependent killing.
CMT93 cells were transfected with the indicated siRNAs and RFP or Perforin-2-RFP expression plasmids. Following stimulation with IFN-γ, transfected cells were infected with a Y. pseudotuberculosis …
Figure 8
Cif enhances pathogenicity in vivo.
Survival curves of C57Bl/6 mice inoculated orogastrically with 108 CFU of wild-type Y. pseudotuberculosis or an isogenic cif::aadA mutant. Animals were weighed daily and euthanized if weight loss …
Figure 9
Bacterial inhibitors of neddylation abolish the bactericidal activity of Perforin-2 in vivo.
Survival curves for two different lineages of Mpeg1 +/+, +/−, and −/− mice following orogastric inoculation with 106 CFU of wild-type Y. pseudotuberculosis or an isogenic cif::aadA mutant. (A–C) n = …
Figure 10
Cif diminishes the bactericidal activity of Perforin-2 in vivo.
Representative organ loads of C57Bl/6 × 129X1/SvJ Mpeg1 (A) +/+, (B) +/− and (C) −/− mice infected with Cif+ or Cif− Y. pseudotuberculosis. Animals were sacrificed 10 days after orogastric …
Figure 11 with 1 supplement
Perforin-2 is monoubiquitylated.
Mpeg1 knockout MEFs were transfected with GFP or Perforin-2-GFP expression plasmids and stimulated with IFN-γ in the presence or absence of LPS. Some cultures were also treated with MLN4924, a small …
Figure 11—figure supplement 1
Monoubiquitylation of Perforin-2.
CMT93 cells were transfected with expression plasmids and stimulated with IFN-γ and LPS as indicated. GFP immunoprecipates were then separated by SDS-PAGE and probed with a Perforin-2 antibody. The …
Figure 12
Ubiquitylation determines the subcellular distribution of Perforin-2.
Mpeg1 knockout MEFs were transfected with (A) Perforin-2-RFP or (B) Perforin-2-KQ-RFP expression plasmids, the latter of which carries K-to-Q mutations of conserved lysines in the cytosolic tail of …
Figure 13 with 2 supplements
Cif blocks trafficking of Perforin-2.
Mpeg1 knockout MEFs were transfected with Perforin-2-RFP or Perforin-2-KQ-RFP expression plasmids and stimulated with IFN-γ for 24 hr prior to infection with (A, B) wild-type Y. pseudotuberculosis …
Figure 13—figure supplement 1
Perforin-2 relocalizes from perinuclear to punctate bodies upon ubiquitylation.
CMT93 cells, a murine rectal epithelial cell line, were transfected with murine Perforin-2-RFP or Perforin-2-KQ-RFP expression plasmids and induced with IFN-γ overnight. Cells were then infected …
Figure 13—figure supplement 2
Perforin-2 perinuclear localization in noninfected CMT93s.
CMT93 cells were transfected with (A) murine Perforin-2-RFP or (B) murine Perforin-2-KQ-RFP and induced overnight with IFN-γ. Images were taken on a Leica confocal microscope with a 63× objective.
Figure 14
Monoubiquitylation of Perforin-2 and inhibition by Cif.
(A) CRLs are modular complexes assembled upon cullin scaffolds such as CUL1. F-box proteins such as βTrCP, which we have shown interacts with Perforin-2, provide substrate specificity within the …
Videos
Video 1
Perforin-2-RFP is located in vesicular bodies following stimulation with LPS.
Mpeg1 knockout MEFs were transfected with a Perforin-2-RFP expression plasmid and stimulated overnight with IFN-γ. After stimulation with LPS for 15 min, cells were fixed and counter stained with …
Video 2
Redistribution of Perforin-2 is dependent upon ubiquitylation.
Mpeg1 knockout MEFs were transfected with a Perforin-2-KQ-RFP expression plasmid. This fusion protein cannot be ubiquitylated because it carries three K-to-Q mutations in the carboxy-terminal tail …
