1. Volume 37, Issue 6, Pages 768-783 (March 2010)
A Soluble Form of the Pilus Protein FimA Targets the VDAC-Hexokinase Complex at Mitochondria to Suppress Host Cell Apoptosis 
Sunil K. Sukumaran, Nai Yang Fu, Chua Boon Tin, Kah Fei Wan, San San Lee, Victor C. Yu 
Molecular Cell 
Volume 37, Issue 6, Pages (March 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
K1 Blocks Cell Death of Host Epithelial Cells at the Level of Mitochondria
(A) K1 invades epithelial cells and causes host cell death at a late phase of infection. Upper: HCT116 cells were incubated with indicated bacteria at moi of 10 for 1 hr. The number of adherent and intracellular bacteria was estimated as described in the Experimental Procedures. Error bars indicate SEM. Lower: At the indicated postinfection time, the percentage of cell death in HCT116 cells infected with indicated bacteria was estimated by PI staining. Error bars indicate SEM.
(B) Activation of Bax during infection. At the indicated postinfection time, total lysates of K1- or EPEC-infected HCT116 cells were immunoprecipitated with the conformation-specific Bax antibody, N20, followed by western blotting with the anti-Bax 2D2 antibody. Total lysates were also analyzed by western blotting using the anti-Bax N20 antibody.
(C) K1-infected cells display divergent kinetics in Bax translocation and integration. HCT116 cells at the indicated time points of postinfection were fractionated into heavy membrane (enriched with mitochondria) and cytosolic fractions. Western blot analysis was carried out to monitor the kinetics of Bax translocation and cytochrome c release. Hsp-60 and Actin were used as loading controls for the mitochondrial and cytosolic fractions, respectively. Integration of Bax into mitochondrial membrane was determined by alkali wash assay.
(D) K1 delays cell death of host cells triggered by multiple apoptotic stimuli. After the infection time of 1 hr, K1-infected HCT116 cells were treated with staurosporine (STS) (1 μM), UV (50 mJ/cm2), or TRAIL (100 ng/ml) for the duration indicated, and the percentage of total cell death was monitored by PI staining. Error bars indicate SEM.
(E) K1 renders a short-term blockade of STS-induced Bax integration. Uninfected and K1-infected HCT116 cells were treated with STS (1 μM) for the indicated time periods. Bax translocation, integration, and cytochrome c release were monitored as in (C) (left). A similar analysis was carried out on vector control (VC) or Bcl-xL (myc-Bcl-xL)-expressing HCT116 cells treated with STS (1 μM) (right).
(F–G) Mitochondria isolated from the K1-infected cells are resistant to cytochrome c release. Mitochondria prepared from uninfected or K1-infected (4 hr, p.i.) HCT116 cells was exposed to increasing concentrations of recombinant Bax (F) or 25 μM BH3-peptide of PUMA, Bim, or Bid (G) for 30 min. The mitochondria (Pellet) were spun down, and the supernatant (Sup) and pellet fractions were immunoblotted with anti-cytochrome c and Hsp-60 antibodies (see also Figure S1).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Identification of FimA to Be a Potent Suppressor of Cytochrome c Release
(A) Experimental scheme for preparing the soluble proteins precipitated from the culture supernatants (SPPCS) of bacterial cultures for testing of the presence of suppressor activity of cytochrome c release.
(B) Suppressor activity of cytochrome c release is present in the culture supernatant of K1. Mitochondria prepared from HCT116 cells were treated with SPPCS from K1 or EPEC cultures grown at 37°C, followed by incubation with increasing concentration of recombinant Bax, and spun down. The pellet (Mitochondria) and the supernatant (Sup) fractions were analyzed by western blotting with the indicated antibodies.
(C) Suppressor activity is most active in K1 cultures grown at 37°C. Mitochondria prepared from HCT116 cells were treated with SPPCS from K1 or EPEC cultures grown at the indicated temperatures, followed by incubation with recombinant Bax as in (B).
(D) Alignment of amino acid sequences of the peptide fragments from the soluble FimA. Protein mass fingerprint data was obtained by MALDI-TOF MS analysis. Amino acid sequences of the peptide fragments of FimA are represented in italics. The N-terminal sequence of the soluble FimA was determined by the Edman degradation method and is represented in italics and bold.
(E) FimA is a potent suppressor of Bax-induced cytochrome c release from isolated mitochondria. Mitochondria from HCT116 cells were preincubated with indicated concentration of IVT (in vitro-translated) FimA (upper) or Bcl-xL (lower) for 20 min, followed by treatment with either 50 or 100 nM recombinant Bax for an additional 30 min. The samples were processed and analyzed as in (B).
(F) Characterization of the polyclonal and monoclonal antibodies of FimA; western blot analysis of the lysates of uninfected or K1-infected HCT116 cells as well as the vector and HCT116 cells transiently expressing HA-FimA, using the P2 polyclonal or 1F3 monoclonal FimA antibodies.
(G) FimA is present in the culture supernatant of multiple enteroinvasive bacterial pathogens. Bacterial lysates (20 μg) and SPPCS (20 μg) of K1, Salmonella, Shigella, EPEC, ETEC, or DH5-α were immunoblotted with the indicated anti-FimA antibodies.
(H) FimA is a critical protein in the culture supernatant of K1 responsible for mediating the suppressor effect on cytochrome c release. FimA was immunodepleted from SPPCS of K1 using the P2 antibody. Mitochondria isolated from HCT116 cells were subsequently treated with SPPCS, FimA-depleted SPPCS (SPPCSΔFimA), or FimA-depleted SPPCS supplemented with IVT FimA or heat-inactivated (HI) (100°C, 5 min) IVT FimA for 20 min before being exposed to the indicated concentration of recombinant Bax for additional 30 min. The samples were subsequently processed and analyzed as in (B).
(I) FimA is a critical protein in the culture supernatant of Salmonella and Shigella responsible for mediating the suppressor effect on cytochrome c release. SPPCS of Shigella and Salmonella were subjected to a similar analysis as in (H) (see also Figure S2).
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4. Figure 3
FimA Localizes to Mitochondria of Host Cells Only during the Early Phase of Infection
(A) Ectopically expressed FimA is enriched in mitochondria. Expression plasmid encoding HA-FimA was transiently transfected into HCT116 cells. Expression of FimA in the individual subcellular fractions indicated by organelle-specific markers was assessed by western blotting using the P2 FimA antibody.
(B) FimA localizes to the outer membrane of mitochondria. Mitochondria isolated from HCT116 cells transiently transfected with the plasmid encoding HA-FimA were treated with 5 μg/ml Proteinase K for 20 min, followed by addition of ice-cold PMSF to terminate the reaction. The samples were analyzed by western blotting with indicated antibodies.
(C) GFP-FimA colocalizes with mitochondrial marker. HCT116 cells transfected with N1-pEGFP (GFP-Vect) or N1-pEGFP-FimA (GFP-FimA) were stained with MitoTracker (red) for confocal microscopy analysis.
(D) Schematic illustration of FimA and its mutants and summary of their expression and mitochondrial localization profiles. FimA-FL (full-length), FimA, and its mutants were evaluated for their expression and mitochondrial localization by transient expression in HCT116 cells. “+” denotes mitochondrial localization; “−”denotes no detectable mitochondrial localization; “NE” indicates no protein expression.
(E) A segment of FimA is sufficient mitochondria-targeting function. HCT116 cells transfected with N1-pEGFP or N1-pEGFP-FimA (aa 65–85) were stained with MitoTracker (red) and analyzed by confocal microscopy.
(F) FimA is enriched in mitochondria in cells infected by the enteroinvasive bacteria. HCT116 cells infected with EPEC, K1, Salmonella (Sal), or Shigella (Shig) were harvested at 4 hr postinfection. Subcellular localization of FimA was monitored by organelle-specific markers by western blotting using the P2 FimA antibody.
(G) FimA localizes to mitochondria of infected cells only during the early phase of infection. HCT116 cells infected with K1 were harvested at the indicated postinfection time points. The cytosolic and mitochondrial fractions were prepared and immunoblotted with the indicated antibodies.
(H) Redistribution of FimA from mitochondria to the cytosol precedes the commencement of Bax integration and cytochrome c release during the infection. HCT116 cells infected with K1 were harvested at the indicated postinfection time points. The mitochondrial and cytosolic fractions were prepared and analyzed by western blotting using the indicated antibodies. Alkali wash analysis was carried out to evaluate the membrane integration of Bax (see also Figure S3 and Table S1).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4
FimA Alone Is Capable of Mediating the Delay of Bax Integration and Apoptotic Death Triggered by Multiple Stimuli
(A) Mitochondria isolated from cells stably expressing FimA are resistant to Bax-induced cytochrome c release. Mitochondria isolated from Vector, HA-FimA, or K1-infected (4 hr, p.i.) HCT116 cells were incubated with indicated concentrations of recombinant Bax for 30 min. The supernatant (Sup) and pellet fractions were analyzed by immunoblotting with the indicated antibodies.
(B) Cell death triggered by apoptotic stimuli is delayed in cells stably expressing FimA. Vector, HA-FimA, or K1-infected HCT116 cells were exposed to the indicated stimuli for the indicated time periods, followed by PI staining to quantify the extent of cell death. Error bars indicate SEM.
(C) Apoptosis induction coincides with dissociation of FimA from mitochondria to cytosol. The FimA-expressing or K1-infected HCT116 cells were treated with STS for the indicated time periods. The cytosol and mitochondria were prepared and analyzed by western blotting with the indicated antibodies.
(D) STS-mediated Bax integration is delayed in the FimA-expressing cells. Mitochondria prepared from FimA-expressing or K1-infected HCT116 cells treated with STS for the indicated time periods were subjected to alkali wash followed by western blot analysis using the N-20 Bax antibody. VDAC1 was probed as a control for integrated mitochondrial protein (see also Figure S4).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 5
Targeting of FimA to Mitochondria Is a Prerequisite Step for K1 to Inhibit Host Cell Apoptosis
(A) The defects in adherence and invasion of K1ΔfimA can be rescued by the alanine scanning mutants of FimA-FL. Adherence and invasion assays were carried out in HCT116 cells with wild-type K1, K1ΔfimA, or K1ΔfimA strains complimented with FimA-FL or the indicated FimA-FL alanine mutants.
(B) Localization of FimA and alanine scanning mutants of FimA. HCT116 cells were infected with WT K1, K1ΔfimA, or the K1ΔfimA strains harboring the indicated FimA-FL. Alanine mutants were harvested at 4 hr postinfection. Total lysates, cytosol, and mitochondria fractions were analyzed by western blotting using the P2 FimA antibody.
(C) Mitochondria association function of FimA is required for K1 to suppress host cell apoptosis. HCT116 cells were infected by WT K1 or K1ΔfimA strains harboring FimA-FL or its indicated alanine substitution mutants. Cell death was measured by PI staining at the indicated postinfection time. Error bars indicate SEM.
(D) Mitochondria association function of FimA is required for K1 to delay Bax integration in host cells. HCT116 cells infected with indicated bacteria were fractionated into mitochondria and cytosolic fractions at the indicated postinfection time. An aliquot of the mitochondrial fraction was used for alkali wash assay to assess the extent of Bax integration. Western blot analysis of the cytosol and mitochondria was carried out to detect the mitochondrial translocation of Bax and the release of cytochrome c during infection using the N-20 Bax and cytochrome c antibodies (see also Figure S5).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 6
FimA Associates with VDAC1 and Strengthens Its Association with HK
(A) FimA associates with VDAC1 during the early phase of infection. HCT116 cells infected with K1 were harvested at the indicated postinfection time points. Equivalent amount of total lysates were immunoprecipitated with the P2 FimA antibody, followed by western blotting with the 3C6 FimA antibody or the indicated VDAC isoform-specific antibodies. Total lysates were also analyzed by western blotting using the P2 and the indicated VDAC isoform-specific antibodies. Mitochondrial fractions were analyzed by western blotting using the indicated antibodies.
(B) Time course of dissociation of HK from VDAC1 coincides with that of initiation of cytochrome c release in both K1- and EPEC-infected cells. HCT116 cells infected with K1 or EPEC were harvested at the indicated postinfection time points. Equivalent amounts of total lysates were immunoprecipitated with anti-VDAC1 antibody, followed by western blotting with the HK (HK-II), 3C6 FimA, or VDAC1 antibody. Total lysates and mitochondrial fractions were also analyzed by western blotting with the indicated antibodies.
(C) VDAC1-bound HK is resistant to displacement by the HK peptide in mitochondria from the K1-infected cells. Mitochondria prepared from uninfected or K1-infected (4 hr, p.i.) HCT116 cells were treated with indicated concentrations of the peptide HXK2VBD for 20 min. The mitochondria were spun down and lysed, and an equivalent amount of lysates were subjected to IP with VDAC1 antibody. The immunoprecipitates were then analyzed by western blotting using the indicated antibodies.
(D) FimA strengthens the association of HK to VDAC1. Mitochondria prepared from HCT116 cells were incubated with IVT FimA (10 nM) or equal volume of rabbit reticulocyte lysates (Control) for 20 min, followed by addition of HXK2VBD to achieve the indicated concentrations. After an additional 20 min incubation time, samples were analyzed as described in (C) (see also Figure S6 and Table S2).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
VDAC1 and HK Are Both Necessary for Mediating the Antiapoptotic Activity of FimA
(A) FimA is significantly less active in binding mitochondria from the VDAC1-deficient cells. Mitochondria prepared from HCT116 cells stably expressing shRNA-3-m or shRNA-3 were incubated with IVT-FimA (10 nM) for 20 min before being subjected to three washes with PBS. The extent of binding of FimA was then determined by western blotting using the P2 FimA antibody.
(B) FimA is ineffective in suppressing Bax-mediated cytochrome c release in isolated mitochondria from the VDAC1 knockdown cells. Mitochondria prepared from HCT116 cells stably expressing shRNA-3-m or shRNA-3 were incubated with IVT-FimA (10 nM) or an equal volume of rabbit reticulocyte lysates (Control) for 20 min before adding recombinant Bax to achieve the indicated concentration. The samples were incubated for an additional 30 min before having the mitochondria spun down, and the supernatant (Sup) and pellet fractions were immunoblotted using cytochrome c and Hsp-60 antibody, respectively.
(C) K1 infection triggers rapid release of cytochrome c in the VDAC1 knockdown cells. HCT116 cells stably expressing shRNA-3-m or shRNA-3 were infected with K1, and the infected cells were harvested at the indicated postinfection time points. Mitochondria were prepared from the infected cells and analyzed by western blotting using the indicated antibodies.
(D) K1 infection induces rapid Bax integration and cytochrome c release in the HK knockdown cells. HCT116 cells were transfected with plasmids encoding HK-I shRNA-6 plus HK-II shRNA-4 or plasmids encoding the mutant shRNAs, HK-I shRNA-6-m plus HK-II shRNA-4-m. HK knockdown cells were infected with K1, and the infected cells were harvested at the indicated postinfection time points. Equivalent amounts of total lysates were immunoprecipitated with anti-VDAC1 antibody, followed by western blotting with the HK (HK-I, -II), 3C6 FimA, or VDAC1 antibody. Total lysates and heavy membrane fractions (mitochondria) were also analyzed by western blotting with the indicated antibodies. Alkali wash analysis was carried out to evaluate membrane integration of Bax.
(E) FimA fails to delay Bax integration and cytochrome c release triggered by STS in the HK knockdown cells. HCT116 cells stably expressing FimA were transfected with the shRNAs and mutant shRNAs as described in (D). HK knockdown cells were treated with STS for the indicated time points. The samples were subsequently processed and analyzed as in (D) (see also Figure S7).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions