Fig 1.
The BepCBhe FIC domain but not a conserved FIC motif or flap region is required for actin stress fiber formation in B. henselae-infected HUVECs.
(A) Schematic view of BepCBhe wild-type and mutant variants analyzed in this figure. The positively charged tail at the C-terminus is represented by +++. The N-terminally fused FLAG-tag triple copy (3xFLAG) is not shown. (B) HUVECs were infected at a multiplicity of infection (MOI) of 400 with isogenic Bhe ΔbepA-G strains expressing FLAG-tagged BepCBhe wild-type or mutant versions, or carrying the empty plasmid. After 48 h of infection, cells were fixed and immunocytochemically stained, followed by fluorescence microscopy analysis. F-actin is represented in green, DNA in blue, and bacteria in red (scale bar = 50 μm). Arrows point to cell fragments resulting from distorted rear end retraction of migrating HUVEC. (C) Expression of 3xFLAG-tagged proteins was analyzed in bacterial lysates of indicated strains by immunoblot (IB) with an anti-FLAG antibody. (D) Cell fragmentation was quantified by manually analyzing 18 images, each containing around 100 cells, per condition. The graph shows the number of cell fragments per 100 cells. Shown are representative results from three independent experiments. BepCBhe**** = BepCBhe H146A, K150A, R154A, R157A; BepCBhe (Flap BepABhe) = BepCBhe A90E, R92K, P93R, K94T, H96W, R97K, V98N, P99A.
Fig 2.
The BepCBhe FIC domain but not a conserved FIC motif or flap region is required for actin stress fiber formation in B. henselae-infected HeLa cells.
(A) HeLa cells were infected with isogenic Bhe ΔbepA-G strains expressing 3xFLAG-tagged BepCBhe wild-type or mutant versions or carrying the empty plasmid at a multiplicity of infection (MOI) of 400. After 48 h of infection, cells were fixed and immunocytochemically stained, followed by fluorescence microscopy analysis. F-actin is represented in green, DNA in blue, and bacteria in red (scale bar = 50 μm). (B) HeLa cells were infected with the same strains as in (A) at MOI 50, 100, 200, 400, and 800, fixed after 24 h and 48 h and stained for F-actin. The graphs show the relative mean fluorescence intensity of the F-actin signal at 24 h (left panel) and 48 h (right panel) infection for the indicated MOIs normalized to the uninfected control. Shown are representative results from three independent experiments. BepCBhe**** = BepCBhe H146A, K150A, R154A, R157A; BepCBhe (Flap BepABhe) = BepCBhe A90E, R92K, P93R, K94T, H96W, R97K, V98N, P99A.
Fig 3.
Both FIC and BID domains are required for BepCBhe–triggered actin stress fiber formation upon ectopic effector expression in HeLa cells.
(A) Schematic view of BepCBhe wild-type and mutant variants analyzed in this figure. The positively charged tail at the C-terminus is represented by +++. The N-terminally fused triple FLAG-tag (3xFLAG) is not shown. (B) HeLa cells were transfected with the indicated plasmids for expression of 3xFLAG-tagged BepCBhe wild-type, mutant versions, or truncations, or no protein as negative control (pEmpty). 24 h after transfection, cells were fixed and immunocytochemically stained, followed by fluorescence microscopy analysis. F-actin is represented in green and DNA in blue (scale bar = 50 μm). (C) Expression of FLAG-tagged proteins was analysed in cell lysates by immunoblot with an anti-FLAG antibody. (D) The mean fluorescence intensity of F-actin shown for conditions shown in (B) was quantified for 74 imaged sites using CellProfiler. Data are represented as dot plots with each data point corresponding to the average of all mean cell intensity values within one imaged site. Statistical significance was determined using Kruskal-Wallis test (**** corresponds to p-value ≤ 0.0001). BepCBhe**** = BepCBhe H146A, K150A, R154A, R157A.
Fig 4.
BepCBhe binds to GEF-H1 and MRCKα.
(A) HeLa cells were infected with Bhe ΔbepA-G expressing FLAG-tagged BepCBhe, or carrying the empty plasmid as a negative control, at MOI of 200. After 24 h of infection, cells were lysed and the lysate incubated in presence of anti-FLAG antibody. 3xFLAG-BepCBhe and interacting proteins were pulled-down with protein G agarose beads and bound proteins were released with SDS-containing buffer. Samples (technical triplicates) were analyzed by mass spectrometry and data obtained for 3xFLAG-BepCBhe and the negative control were compared (see S1 Table for a listing of all identified proteins). (B) Proposed model of BepC-triggered actin stress fiber formation with reference to the experimental data presented for validation. (C) HUVECs were infected with Bhe ΔbepA-G expressing 3xFLAG-tagged BepCBhe or carrying empty plasmid at MOI of 200 for 24 h. Cells were lysed and incubated in presence of anti-GEF-H1 antibody or anti-MRCKα antibody. Antibody-bound proteins were subsequently pulled-down with protein G agarose beads, followed by elution with SDS-containing buffer. Cell lysates before pull-down and pull-down samples were analyzed by immunoblot using antibodies against FLAG-tag, GEF-H1, or MRCKα. (D) HeLa wild-type or knocked-out cells for GEF-H1 or MRCKα were infected with Bhe ΔbepA-G expressing FLAG-tagged BepCBhe or carrying the empty plasmid as a negative control at MOI of 200. After 24 h of infection, cells were lysed and incubated with anti-FLAG antibodies. 3xFLAG-BepCBhe was pulled-down with protein G agarose beads before eluted with SDS. Cell lysates before pull-down and pull-down samples were analyzed by immunoblot against FLAG-tag, GEF-H1, or MRCKα. (E) Pull-down fractions of three independent experiments samples as shown in (D) were quantified using ImageJ and plotted as relative intensities of the bands normalized to the wild-type control. Shown are representative results from three independent experiments.
Fig 5.
GEF-H1 is essential for BepCBhe–triggered actin stress fiber formation while MRCKα is dispensable.
(A) Proposed model of BepC-triggered actin stress fiber formation with indication of the GEF-H1 and MRCKα knock-out. (B) HeLa cells were co-transfected with two different plasmids encoding Cas9 and a sgRNA, specific either to the first or the last exon of the target gene (GEF-H1 or MRCKα). After selection and expansion of transfected cells, expression of GEF-H1 or MRCKα was tested by immunoblot analysis. Tubulin was used as loading control. (C-E) HeLa cells wild-type, GEF-H1 KO, and MRCKα KO were infected with Bhe ΔbepA-G expressing FLAG-tagged BepCBhe, or carrying the empty plasmid as a negative control, at MOI of 400. After 48 h of infection, cells were fixed, stained by immunocytochemistry and analyzed by fluorescence microscopy. (C) F-actin is represented in green, DNA in blue, and bacteria in red. (D) The mean fluorescence intensity of F-actin for conditions shown in (C) was quantified for 111 imaged sites using CellProfiler. Data are represented as dot plots with each data point corresponding to the average of all mean cell intensity values within one imaged site normalized to the uninfected wild-type (WT) control. Statistical significance was determined using Kruskal-Wallis test (**** corresponds to p-value ≤ 0.0001). (E) Anti-GEF-H1 staining is represented in white (scale bar = 50 μm). Shown are representative results from three independent experiments.
Fig 6.
BepCBhe recruits eGFP-GEF-H1 to the plasma membrane via binding of the FIC-OB domain to eGFP-GEF-H1 and binding of the BID domain to the plasma membrane shown by immunocytochemistry.
(A-B) HeLa cells were co-transfected with an expression plasmid for eGFP-GEF-H1 and the indicated plasmids for either (A) no expression or (B) expression of either 3xFLAG-BepCBhe, 3xFLAG-BepCBhe(FIC-OB), or 3xFLAG-BepCBhe(OB-BID). After 24 h, cells were fixed and stained by immunofluorescence labeling for FLAG and microtubule before being analyzed by fluorescence microscopy (scale bar = 25 μm). The x-z sections presented correspond to orthogonal cuts at the white lines displayed in the microtubule channel.
Fig 7.
BepCBhe recruits GEF-H1 to the plasma membrane via binding of the FIC-OB domain to GEF-H1 shown by pull-down and binding of the BID domain to the plasma membrane shown by subcellular fractionation.
(A) HeLa cells were co-transfected with an expression plasmid for eGFP-GEF-H1 or eGFP and the indicated plasmids for expression of either 3xFLAG-BepCBhe, 3xFLAG-BepCBhe(FIC-OB), or 3xFLAG-BepCBhe(OB-BID). After 24 h, cell lysates were prepared and used for a FLAG pull-down assay. Bound proteins were analyzed by immunoblot with anti-FLAG, anti-GEF-H1 and anti-GFP antibodies. The signal visible in the anti-GFP blot for the marker lane was probably due to unspecific cross-reactivity. (B) HeLa cells were transfected with 3xFLAG-BepCBhe, 3xFLAG-BepCBhe(FIC-OB), or 3xFLAG-BepCBhe(OB-BID) for 24 h, then cell lysates were prepared, separated into membrane and cytosolic fractions and analyzed by immunoblot with anti-FLAG and anti-GEF-H1 antibodies. Anti-tubulin and anti-Na/K ATPase antibodies were used as cytosolic and membrane markers, respectively. Shown are representative results from three independent experiments.
Fig 8.
Model of BepC-triggered actin stress fibers formation mediated by the recruitment of GEF-H1 to the plasma membrane.
Upon translocation, BepC localizes to the plasma membrane via its BID domain and binds GEF-H1 via its FIC-OB domains. There, GEF-H1 activates RhoA by exchanging GDP for GTP, allowing activation of the downstream kinase ROCK. ROCK-dependent phosphorylation of myosin light chain (MLC) will then induce actin stress fibers formation.