BepC triggers actin stress fiber formation and cell fragmentation in human umbilical vein endothelial cells

Infection with Bhe ΔbepC_Bhe deletion mutants and ectopic expression of mCherry-BepC_Bhe in HUVECs implicated BepC in triggering actin stress fiber formation and the linked cell fragmentation phenotype resulting from distorted rear-end detachment during cell migration [11]. To demonstrate that these prominent phenotypes can result from VirB/VirD4-dependent translocation of BepC_Bhe into infected HUVECs, we expressed BepC_Bhe with a N-terminal triple FLAG (Fig 1A) in the effector-free background of the Bhe ΔbepA-G strain [8]. Translocation of 3xFLAG-BepC_Bhe by this strain triggered both F-actin-dependent phenotypes in dependency of infection time and multiplicity of infection (MOI) (Figs 1B and S1A), while the isogenic control strain containing the empty expression plasmid did not noticeably alter the F-actin cytoskeleton compared to uninfected cells (Figs 1B, S1A and S1B). BepC_Bhe-dependent actin stress fiber formation was evident at MOI of 200 at 24 h, while at this time-point cell fragmentation became only visible at MOI of 400. Generally, stress fiber formation and cell fragmentation phenotypes were more pronounced at 48 h than at 24 h, and this late time-point was thus used for most follow-up experiments. Of note, BepC-triggered cell fragmentation resulted in decreased cell number, while cellular fragments did not display morphological features of apoptosis (i.e., blebbing) or necrotic cell death (i.e., lysis).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. The BepC_Bhe 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 BepC_Bhe 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 BepC_Bhe 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. BepC_Bhe**** = BepC_Bhe H146A, K150A, R154A, R157A; BepC_Bhe (Flap BepA_Bhe) = BepC_Bhe A90E, R92K, P93R, K94T, H96W, R97K, V98N, P99A.

https://doi.org/10.1371/journal.ppat.1008548.g001

The slow kinetics and high MOI-dependency of BepC_Bhe–triggered stress fiber formation suggest that the effector may act on its host target(s) by protein-protein interaction (e.g., by target sequestration or mislocalization) rather than by an enzymatic mechanism. While BepC contains a well-conserved FIC domain that typically catalyzes an enzymatic activity such as AMPylation [20], its FIC motif (HxFxKGNGRxxR) differs from the canonical FIC motif (HxFxD/EGNGRxxR), which is defined by essential amino acids in the active site of these AMP-transferases, in one amino acid position (D/E replaced by K) [16]. Despite this single amino acid exchange, the FIC domain of BepC might still harbor enzymatic activity; possibly one that differs from AMPylation. However, we reasoned that the mutations of this lysine and of three additional amino acids known to be essential for enzymatic activity in FIC domains with a canonical FIC motif [20,21,28] should incapacitate any presumable enzymatic activity of the BepC FIC domain. We thus constructed the quadruple mutant BepC_Bhe**** with the amino acid exchanges H146A, K150A, R154A, R157A, resulting in a highly degenerated FIC motif (AxFxAGNGAxxA, Fig 1A). Moreover, we constructed another mutant that might compromise BepC-specific target modification by exchanging the flap region, which registers the target protein to the active site of FIC domains, between BepC_Bhe and BepA_Bhe (3xFLAG-BepC_Bhe(Flap BepA_Bhe), Fig 1A). Both of these BepC_Bhe mutant proteins maintained the same capacity to trigger stress fiber formation and cell fragmentation as BepC_Bhe, indicating that BepC_Bhe may not require enzymatic modification of host targets to trigger actin rearrangements (Fig 1B and 1D). However, deletion of the FIC domain (3xFLAG-BepC_Bhe(OB-BID), Fig 1A) rendered the truncated BepC_Bhe protein unable to produce any phenotype (Fig 1B and 1D), despite being expressed as the same level as the wild-type and mutants versions of BepC_Bhe (Fig 1C).

In summary, the FIC domain of BepC is required for actin stress fiber formation and cell fragmentation, but neither a conserved FIC motif nor a specific flap region are necessary, suggesting that the actin phenotype is linked to a non-enzymatic activity.

BepC-triggered actin stress fiber formation in HeLa cells is dependent on both the FIC and the BID domain

To facilitate further cellular and molecular analysis of the mechanism underlying BepC-triggered actin stress fiber formation in an established cell line, we adopted the previously published HeLa cells infection model [25]. The capacity of isogenic strains expressing BepC_Bhe wild-type and corresponding mutant variants to trigger actin stress fiber formation in HUVEC was fully reproduced in HeLa cells (Fig 2A and 2B). Additionally, staining infected HeLa cells with an anti-FLAG antibody confirmed the translocation of all BepC_Bhe variants, excluding a translocation defect of BepC_Bhe (OB-BID) (S2A Fig). The absence of actin stress fiber formation in HeLa cells infected with the translocation-deficient strain Bhe ΔbepA-G, ΔvirB4 expressing BepC definitely confirmed the T4SS dependency of the actin phenotype (S3 Fig).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. The BepC_Bhe 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 BepC_Bhe 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. BepC_Bhe**** = BepC_Bhe H146A, K150A, R154A, R157A; BepC_Bhe (Flap BepA_Bhe) = BepC_Bhe A90E, R92K, P93R, K94T, H96W, R97K, V98N, P99A.

https://doi.org/10.1371/journal.ppat.1008548.g002

As a complementary approach to VirB/VirD4-dependent effector translocation, we tested how ectopic expression of BepC_Bhe wild-type and mutant variants affected actin stress fiber formation (Fig 3A). The phenotypic data and their quantification obtained for ectopic expression in HeLa cells (Fig 3B and 3D) are in full agreement with translocation-dependent infection phenotypes in both HUVEC and HeLa (Figs 1B, 2A and 2B). Importantly, the ectopic expression approach allowed us to test also a C-terminal truncation resulting in deletion of the entire BID domain and positively charged tail sequence (3xFLAG-BepC_Bhe(FIC-OB), Fig 3A) that could not be tested in the infection assay as deletion of this bipartite secretion signal abolishes VirB/VirD4-dependent protein translocation [8]. While being expressed at similar level to the wild-type effector (Figs 3C, and S2B), the lack of increased actin stress fiber formation by ectopic expression of this C-terminal truncation demonstrated the essential role of the BID domain in mediating this phenotype (Fig 3B and 3D).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Both FIC and BID domains are required for BepC_Bhe–triggered actin stress fiber formation upon ectopic effector expression in HeLa cells.

(A) Schematic view of BepC_Bhe 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 BepC_Bhe 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). BepC_Bhe**** = BepC_Bhe H146A, K150A, R154A, R157A.

https://doi.org/10.1371/journal.ppat.1008548.g003

The high level of sequence conservation of BepC homologs in different Bartonella species is indicative of a conserved molecular function [16]. We thus tested whether the capacity of BepC_Bhe to trigger actin stress fiber formation is conserved among BepC homologs. HeLa cells were infected with Bhe ΔbepA-G expressing FLAG-tagged BepC of Bhe, B. quintana (Bqu), B. tribocorum (Btr), B. taylorii (Bta), or B. grahamii (Bgr). Increased F-actin stress fiber formation was evident for all BepC homologs, except for BepC_Bgr that was highly similar to the negative control strain Bhe ΔbepA-G pEmpty (S4A and S4C Fig), likely as the result of the low expression level (S4B Fig). However, ectopic expression of these natural BepC variants demonstrated strongly increased stress fiber formation for each of them, including BepC_Bgr (S4D, S4E and S4F Fig), suggesting that the lack of phenotype for BepC_Bgr in the infection assay was indeed a false-negative result.

In summary, our data demonstrate that both FIC and BID domains are required for BepC-triggered actin stress fiber formation. Moreover, these actin rearrangements are triggered by all tested BepC homologs from various Bartonella species, suggesting that this conserved effector function targeting the actin cytoskeleton is likely playing a crucial role during Bartonella infection.

GEF-H1 and MRCKα form a complex that co-immunoprecipitates with BepC

In order to search for potential host targets of BepC_Bhe, we identified interacting host proteins by an interactomics approach. To this end, we infected HeLa cells with Bhe ΔbepA-G expressing triple FLAG-tagged BepC_Bhe or the isogenic strain containing the empty expression plasmid. Following cell lysis, 3xFLAG-BepC_Bhe was pulled down with anti-FLAG-tag antibodies and co-immunoprecipitating host proteins were identified by mass spectrometry (Fig 4A and S1 Table). Compared to the negative control, six proteins showed an increase of at least 8-fold and a q-value lower than 0.05 (Fig 4A). Three of these six proteins were identified by a single peptide. These proteins were not followed up as their relevance was uncertain. As expected, one of the three remaining proteins corresponded to BepC_Bhe as the bait. The two outstanding interactors, GEF-H1 and MRCKα, were particularly interesting as both are involved in regulating F-actin rearrangements (Fig 4B). On one hand, GEF-H1 promotes the activation of RhoA by exchanging GDP for GTP, which then via ROCK activation and subsequent phosphorylation of myosin light chain (MLC) ultimately leads to actin stress fiber formation [28–31]. On the other hand, MRCKα is a downstream effector of the Cdc42 pathway and directly phosphorylates MLC and, as ROCK, inhibits the myosin light chain phosphatase (MLCP), thereby promoting actin stress fiber formation as well [29–31].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. BepC_Bhe binds to GEF-H1 and MRCKα.

(A) HeLa cells were infected with Bhe ΔbepA-G expressing FLAG-tagged BepC_Bhe, 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-BepC_Bhe 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-BepC_Bhe 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 BepC_Bhe 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 BepC_Bhe 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-BepC_Bhe 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.

https://doi.org/10.1371/journal.ppat.1008548.g004

To validate the BepC_Bhe interaction with GEF-H1 and MRCKα as identified by interactomics in HeLa cells, we performed a reciprocal co-immunoprecipitation experiment in a different cell type. To this end, HUVECs were infected with Bhe ΔbepA-G expressing FLAG-tagged BepC_Bhe or carrying the empty expression plasmid. Following pull-down of GEF-H1 or MRCKα (Fig 4C), 3xFLAG-BepC_Bhe was detected in both pull-down fractions, confirming that the three proteins are part of a complex. In contrast, 3xFLAG-BepC_Bhe (OB-BID) did not co-immunoprecipitate with either GEF-H1 or MRCKα, indicating that the FIC domain is critical for this interaction. Moreover, even in the absence of BepC_Bhe, GEF-H1 co-immunoprecipitated with MRCKα and vice versa, indicating that MRCKα and GEF-H1 are part of a native complex that to our knowledge has not been described yet (Fig 4C).

In summary, interactomics identified a native complex of GEF-H1 and MRCKα that co-immunoprecipitated with BepC_Bhe, leaving it open whether BepC interacts in a direct manner with GEF-H1, or with MRCKα, or with both.

The interaction of GEF-H1 with BepC is independent of MRCKα

To untangle the molecular interactions within the complex of BepC, GEF-H1, and MRCKα, we generated HeLa knock-out cell lines for GEF-H1 or MRCKα by CRISPR/Cas9 gene editing. To this end, we co-transfected HeLa cells with two different plasmids encoding Cas9 and sgRNAs that are specific to either the first or the last exon of the gene of interest. Following selection and polyclonal expansion of knock-out cells, we tested expression of GEF-H1 or MRCKα via immunoblot analysis. The data indicate a complete knock-out for MRCKα, and a partial knock-out for GEF-H1 with a nevertheless strongly diminished protein level compared to the parental wild-type cell line (Figs 4D and 5B).

The established knock-out cell lines for GEF-H1 or MRCKα and the parental wild-type cell line were then used to unravel the interaction between these cellular proteins and BepC by using infection and co-immunoprecipitation analysis. To this end, the two knock-out and the parental cell lines were infected with Bhe ΔbepA-G expressing FLAG-tagged BepC_Bhe or carrying the empty expression plasmid. Following cell lysis, 3xFLAG-BepC_Bhe was pulled-down and tested for co-immunoprecipitation of GEF-H1 and/or MRCKα by immunoblot analysis (Fig 4D). GEF-H1 interaction with 3xFLAG-BepC_Bhe was indistinguishable for wild-type cells and MRCKα knock-out cells, indicating that the interaction is independent of MRCKα (Fig 4D, pull-down and Fig 4E). In contrast, MRCKα interaction with 3xFLAG-BepC_Bhe was reduced by about 60% in the partial GEF-H1 knock-out cell line compared to wild-type cells (Fig 4D, pull-down and Fig 4E), suggesting that this interaction is at least in part dependent on GEF-H1. Clarification of this finding may require the establishment of a complete GEF-H1 knock-out cell line, which, however, may not be viable.

Overall, our data indicate that the interaction of BepC_Bhe with GEF-H1 is independent of MRCKα, while the interaction with MRCKα depends at least partially on GEF-H1.

BepC–triggered actin stress fiber formation requires GEF-H1 and is associated with GEF-H1 relocalization

Next, we used the established GEF-H1 or MRCKα knock-out cells and parental wild-type cells in infection experiments to test for the roles of GEF-H1 and MRCKα in mediating BepC_Bhe–triggered stress fiber formation (Fig 5A). To this end, we infected these three cell lines with Bhe ΔbepA-G expressing FLAG-tagged BepC_Bhe or carrying the empty expression plasmid, or left them uninfected as an additional negative control. Staining for F-actin unequivocally demonstrated that GEF-H1 is essential for mediating BepC_Bhe–triggered stress fiber formation, while MRCKα is neglectable for this process (Fig 5C and 5D). Strikingly, staining with anti-GEF-H1 antibodies provided first evidence for a BepC–dependent relocalization of GEF-H1 (Fig 5E). The GEF-H1 knock-out cells displayed invariant background staining for all three conditions. However, parental wild-type and MRCKα knock-out cells displayed a characteristic cytoplasmic GEF-H1 staining pattern consistent with microtubular association in both uninfected and control infection conditions. In contrast, GEF-H1 seemed to relocalize to the plasma membrane upon infection with the FLAG-tagged BepC_Bhe expressing strain (Fig 5E).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. GEF-H1 is essential for BepC_Bhe–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 BepC_Bhe, 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.

https://doi.org/10.1371/journal.ppat.1008548.g005

In summary, we demonstrated an essential role of GEF-H1 for BepC_Bhe–dependent actin stress fiber formation and provided first indications that BepC_Bhe translocation mediates a relocalization of GEF-H1 from a canonical microtubular-association to a putative plasma membrane localization.

BepC interacts with GEF-H1 via its FIC domain while plasma membrane association is mediated by the BID domain

To substantiate our findings on a BepC-dependent relocalization of GEF-H1 from microtubules to the plasma membrane and to determine which BepC domain interactions are involved in mediating this effect, we have used an ectopic co-expression approach in HeLa cells. To follow GEF-H1 localization, we expressed a functional eGFP-GEF-H1 fusion that was previously reported to localize primarily to microtubules, indistinguishably from endogenous GEF-H1 [32]. Co-transfection with an empty expression plasmid (pEmpty) confirmed this canonical staining pattern as demonstrated by colocalization with microtubules (Fig 6A). However, co-expression with Flag-tagged BepC_Bhe showed that, while a part of the GEF-H1 signal remained associated with microtubules, a significant proportion of the eGFP-GEF-H1 signal co-localized with 3xFLAG-BepC_Bhe at the plasma membrane (Fig 6B, upper panel of x-y projections and left panel of x-z sections). In sharp contrast, the microtubule-associated localization of eGFP-GEF-H1 was unperturbed when co-expressed with either 3xFLAG-BepC_Bhe(FIC-OB) or 3xFLAG-BepC_Bhe(OB-BID) truncation constructs (Fig 6B, middle or lower panel of x-y projections or middle or right panel of x-z sections, respectively). Strikingly, 3xFLAG-BepC_Bhe(FIC-OB) colocalized with both eGFP-GEF-H1 and microtubules (Fig 6B, middle panels of x-y projections and x-z sections), indicating that the soluble FIC-OB fragment binds to GEF-H1 without dissociating it from microtubules. On the contrary, 3xFLAG-BepC_Bhe(OB-BID) displayed a plasma membrane localization without any sign of co-localization with microtubule-bound eGFP-GEF-H1 (Fig 6B, lower panel of x-y projections and right panel of x-z sections).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. BepC_Bhe 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-BepC_Bhe, 3xFLAG-BepC_Bhe(FIC-OB), or 3xFLAG-BepC_Bhe(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.

https://doi.org/10.1371/journal.ppat.1008548.g006

To further corroborate these findings on BepC domain-specific interaction with GEF-H1 or association to the plasma membrane derived by fluorescent microscopy, we performed complementary pull-down assays and subcellular fractionation analysis (Fig 7). By pulling-down 3xFLAG-tagged BepC_Bhe or the FIC-OB or OB-BID truncation derivatives in Hela cells, we could demonstrate that endogenous GEF-H1 preferentially co-immunoprecipitate with the full-length BepC_Bhe, despite being less expressed. Additionally, BepC_Bhe FIC-OB also showed a discrete but apparent binding of endogenous GEF-H1 while BepC_Bhe OB-BID construct did not show any interaction (Fig 7A, right half of immunoblots). A corresponding pull-down experiment with HeLa cells ectopically expressing eGFP-GEF-H1 confirmed the findings with endogenous GEF-H1 by showing co-immunoprecipitation of eGFP-GEF-H1 with full-length BepC_Bhe and FIC-OB constructs, while in comparison the OB-BID construct displayed only minute amounts of co-immunoprecipitating eGFP-GEF-H1 (Fig 7A, left half of immunoblots), demonstrating that ectopically expressed eGFP-GEF-H1, as also used in the microscopic analysis in Fig 6, behaves similarly than endogenous GEF-H1 regarding the specific interaction with BepC_Bhe via its FIC domain. Next, we used subcellular fractionation to test for the localization of 3xFLAG-tagged BepC_Bhe or its FIC-OB or OB-BID truncation derivatives. HeLa cells expressing either one of these constructs were ruptured and the full lysate was fractionated in the cytosolic and membrane fraction by ultracentrifugation. Fig 7B shows that the BepC_Bhe full-length construct was entirely fractionating with membranes. The OB-BID construct was as well found primarily in the membrane fraction with only minute amounts in the cytosol, while in sharp contrast the FIC-OB construct was predominately present in the cytoplasmic fraction with scarce amounts fractioning to membranes. Due to the disassembly of microtubuli during cell rupture, GEF-H1 was found to localize predominately to the membrane fraction, which did not allow to test for its localization based on interaction with the three BepC_Bhe constructs.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. BepC_Bhe 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-BepC_Bhe, 3xFLAG-BepC_Bhe(FIC-OB), or 3xFLAG-BepC_Bhe(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-BepC_Bhe, 3xFLAG-BepC_Bhe(FIC-OB), or 3xFLAG-BepC_Bhe(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.

https://doi.org/10.1371/journal.ppat.1008548.g007

Taken together, the orthogonal data obtained from three different assays are fully consistent in indicating that the FIC domain and possibly the OB fold is required for BepC binding to GEF-H1, while the BID domain and possibly the OB fold is necessary for plasma membrane interaction. Together, we conclude that these domain interactions mediate the BepC-dependent relocalization of GEF-H1 from the canonical microtubule-association to the plasma membrane.

BepC-triggers actin stress fiber formation via the GEF-H1/RhoA/ROCK/pMLC pathway

While GEF-H1 binds to microtubules in an inactive conformation it gains GEF activity in association with membranes [32,33], where it activates either RhoA or Rac1 [34]. BepC-mediated recruitment of GEF-H1 to the plasma membrane should thus activate the RhoA and/or Rac1 pathway. Given that the RhoA pathway triggers stress fiber formation, while the Rac1 leads to lamellipodia formation [35], we reasoned that BepC/GEF-H1-mediated stress fiber formation is dependent on the RhoA pathway. To demonstrate this experimentally, we tested the involvement of components of the RhoA pathway in the BepC-triggered phenotype, i.e., by inhibiting RhoA or the Rho-kinase ROCK, and by evaluating the phosphorylation of the ROCK-substrate myosin light chain (pMLC) (S5A and S6A Figs). Rho inhibitor I, which inactivates RhoA, RhoB and RhoC in living cells [36], was found to interfere with stress fiber formation mediated by 3xFLAG-BepC_Bhe in a concentration dependent manner (S5B and S5C Fig). Similarly, Y-27632, which inhibits ROCK in living cells [37], inhibited stress fiber formation by 3xFLAG-BepC_Bhe in a concentration-dependent manner (S5D and S5E Fig). Finally, phosphorylation levels of MLC correlated with stress fiber formation triggered by 3xFLAG-BepC_Bhe and active mutant constructs (S6B and S6C Fig).

Taken together, these data indicate that BepC activates a GEF-H1/RhoA/ROCK/pMLC signaling pathway in order to trigger actin stress fiber formation.

Bacterial strains and growth conditions

The bacterial strains used in this study are listed in S2 Table. Bartonella species were grown on Columbia blood agar (CBA, Oxoid, CM0331) plates containing 5% defibrinated sheep blood (Oxoid, SR0051) at 35°C and 5% CO₂ for 3 days then expended for 2 days on new plates. When necessary, media were supplemented with 30 μg/ml kanamycin, 100 μg/ml streptomycin. E. coli strain was cultivated in Luria-Bertani liquid medium (LB) or on LB agar on plates (LA) at 37°C overnight. Media were supplemented with 50 μg/ml kanamycin and 1 mM diaminopimelic acid (DAP, Sigma, D1377).

The Bhe ΔbepA-G, ΔvirB4 mutant was generated by a two-step gene replacement procedure as described previously [38]. In brief pRS25 [39] was used to generate an in-frame ΔvirB4 deletion in the MSE150 Bhe ΔbepA-G background [8] resulting in the strain LU B2-61.

Construction of plasmids used in this work

Bartonella expression plasmids used in this study are listed in S3 Table. Eukaryotic expression plasmids used in this study are listed in S4 Table. Plasmids construction details as summarized in S5 Table and S6 Table and the used PCR primers as listed in S7 Table.

Conjugation of Bartonella-expression plasmids into Bartonella

Bartonella henselae ΔbepA-G (MSE150) was grown on CBA plates in presence of 100 μg/ml streptomycin at 35°C and 5% CO₂ for 3 days then expanded on new plates for 2 days. The day before conjugation, 5 ml of LB containing 1 mM DAP and 50 μg/ml kanamycin were inoculated with a conjugation strain (JKE170) containing the plasmid of interest. After overnight incubation at 37°C, a subculture was prepared by inoculating 5 ml of LB containing 1 mM DAP and 50 μg/ml kanamycin with 200 μl of overnight culture before being incubated for 2 h at 37°C. In order to remove antibiotics, 500 μl of the subculture was centrifuged for 4 min at 2’000 x g and the bacterial pellet was resuspended in 500 μl of M199 (Gibco, 22340–020) supplemented with 10% of heat-inactivated fetal calf serum (FCS), the washing step was repeated once. The same process was applied to Bartonella, bacteria were harvested in 1 ml of M199 10% heat-inactivated FCS and centrifuged for 4 min at 2’000 x g. The bacterial pellet was resuspended in 500 μl of M199 10% heat-inactivated FCS before being centrifuged again and resuspended in 100 μl of M199 10% heat-inactivated FCS. 20 μl of E. coli were mixed with 100 μl of Bartonella and incubated for 5 h at 35°C, 5% CO₂ on a nitrocellulose filter deposited on a CBA plate supplemented with 1 mM of DAP. The filter was transferred in an Eppendorf tube containing 1 ml of M199 10% heat-inactivated FCS and the bacteria were resuspended by gently shaking. 50 μl were plated on a CBA plate supplemented with 30 μg/ml kanamycin and 100 μg/ml streptomycin. A single colony was selected and subsequently tested to confirm the presence of the plasmid.

Analysis of effector protein expression in B. henselae

B. henselae were grown on CBA plates for three days and expended for 2 days on new plates as described above. To analyze protein expression, bacteria were inoculated at OD 0.1 in 5 ml of M199 containing 10% heat-inactivated FCS with 10 μM IPTG and grown at 35°C and 5% CO₂ for 24 h. Bacteria were harvested by centrifugation, the bacterial pellet was resuspended in SDS-sample buffer to obtain OD 1 and analyzed by immunoblot.

Cell culture

Human umbilical vein endothelial cells (HUVEC) were isolated as described before (Dehio et al., 1997) and cultured at 37°C with 5% CO₂ in Endothelial Cell Growth Medium (ECGM, Promocell, C-22010) supplemented with Endothelial Cell Growth Medium SupplementMix (Promocell, C-39215).

HeLa cells were cultured at 37°C with 5% CO₂ in DMEM (Sigma, D6429) supplemented with 10% heat-inactivated FCS.

Cell infection for microscopy

HUVECs were plated at a density of 3’000 cells/well in a 96-well plate (Corning, #3904) pre-coated with 0.2% of gelatin using supplemented ECGM. HeLa cells were seeded at a density of 12’500 cells/well in a μ-Slide 8 Well (Ibidi, Cat. N°:80826) or at a density of 2’000 cells/well in a 96-well plate (Corning, #3904) using DMEM supplemented with 10% heat-inactivated FCS. The next day, cells were infected with Bartonella at the indicated MOI in M199 (Gibco, 22340–020) supplemented with 10% of heat-inactivated FCS in presence of 10 μM of isopropyl-β-D-thiogalactoside (IPTG, Biochemica, A1008). After incubation at 35°C and 5% CO₂, cells were fixed with 3.7% of paraformaldehyde for 10 minutes and washed 3 times with PBS.

Cell transfection

HeLa cells were seeded at a density of 12’500 cells/well in a μ-Slide 8 Well (Ibidi, Cat. N°:80826) or at a density of 2’000 cells/well on a 96-well plate (Corning, #3904) using DMEM supplemented with 10% of heat-inactivated FCS. The next day, cells were transfected according to manufacturer instruction with a transfection mix containing a ratio of 1 μg of plasmid for 2 μl FuGene HD transfection reagent (Promega, REF E2311) diluted in DMEM without FCS. After transfection for 24 h at 37°C and 5% CO₂, cells were fixed with 3.7% of paraformaldehyde for 10 minutes and washed 3 times with PBS.

Immunostaining

Fixed cells were permeabilized for 10 minutes with PBS 0.2% BSA (Sigma, A9647) and 0.5% Triton X-100 (Sigma, T9284). After being washed 3 times with PBS with 0.2% BSA, cells were incubated overnight at 4°C in the presence of the primary antibody (S8 Table) diluted in PBS with 0.2% BSA. After 2 more washes with PBS with 0.2% BSA, cells were incubated for 2 h in the dark in presence of the secondary antibody (S8 Table), DAPI (Sigma, D9542, 1 μg/ml) and, when indicated, DY-547P1 phalloidin (Dyomics GmbH, final concentration 1/250) diluted in PBS with 0.2% BSA. Cells were finally washed 3 times with PBS. 96-well plates were imaged with an MD ImagXpress Micro automated microscope from Molecular devices and fluorescence was detected at 10x magnification. Images were processed in MetaXpress. For μ-Slide, the stained samples were analyzed using a LEICA point scanning confocal “SP8”microscope (Imaging Core Facility, Biozentrum, University of Basel, Switzerland). Z-stacks with 34–40 focal planes with a spacing of 0.45 μm were recorded and images were reconstructed by Z-projection using ImageJ.

Subcellular fractionation of cell lysates

Subcellular fractionation was based on procedures described before [40] with some modifications. In brief, cells were washed three times with cold PBS, before swelling buffer (50 mM Hepes pH 7.5, 15 mM NaCl, 2mM MgCl2, protease inhibitors) was added. Cells were harvested by scraping, transferred to a 1.5 ml tube and incubated 15 min on ice. Then cells were lysed by sonication (2x 10 pulses, 100% intensity) and incubated again for 15 min on ice. A sample of the resulting cell lysate was kept as full lysate. The remaining sample was fractionated by centrifugation. The nuclei fraction was separated by centrifugation at 800 x g for 15 min at 4°C, the supernatant was further fractionated by centrifugation at 100,000 × g for 1 hour at 4°C into the cytosolic supernatant fraction and the membrane pellet. For analysis by immunoblot, SDS-sample buffer was added. The membrane fraction was resuspended in swelling buffer containing 1% Nonidet P40 substitute (Sigma, 74385) before addition of the sample buffer. To facilitate solubilization, sample were twice heated at 95°C and sonicated. Analysis was performed via immunoblot as described below.

Pull-down assay

HeLa cells or HUVECs were plated in round plates (Falcon, REF 353003) at a density of 365’000 or 544’000 cells per plate, respectively. The cells were then incubated overnight at 37°C with 5% CO₂ in DMEM complemented with 10% heat-inactivated FCS. In the morning, cells have been infected with the indicated strain of Bartonella at MOI of 200 for 24 h at 35°C with 5% CO₂ in M199 supplemented with 10% heat-inactivated FCS in presence of 10 μM of IPTG. After infection, cells were washed 3 times with ice-cold PBS and lysed with lysis buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, protease inhibitor (Roche, 11836170001), and 1% Nonidet P40 substitute (Sigma, 74385). Cell lysates were collected with a cell scraper and incubated 30 minutes on ice. After centrifugation at 20’000 x g for 30 min at 4°C, the supernatants were incubated in presence of 20 μl of protein G agarose beads (Roche, 11243233001) for 3 h at 4°C on a rotor to reduce unspecific binding. After removing the beads by centrifugation for 30 seconds at 12’000 x g, 2 μg of antibody (S8 Table) was added to the supernatant. After 3 h of incubation at 4°C on a rotor, 20 μl of protein G agarose was added to the lysates and incubated overnight at 4°C on a rotor. The next morning, agarose beads were collected by centrifugation for 30 seconds at 12’000 x g before being washed 2 times with lysis buffer and 2 more times with lysis buffer without NP-40. Proteins were eluted from the beads by incubation at 95°C for 10 minutes in SDS sample buffer. Elution fractions and cell lysates before pull-down were analyzed by immunoblot. The same protocol was applied for samples analyzed by mass spectrometry although one cell culture flask of 150 cm2 was used per infection and that proteins were eluted by incubation at 95°C for 10 minutes with 2% SDS.

Sample preparation for mass spectrometry

Proteins were precipitated with trichloroacetic acid and incubated 10 min at 4°C. The protein pellet was washed twice with cold acetone and resuspended with 4 M urea. Then the samples were treated with 5 mM of tris(2-carboxyethyl)phosphine (TCEP) for 30 min at 37°C in order to reduce disulfide bonds. After incubation, iodoacetamide (1.8 mg/ml final) was added to the samples to irreversibly prevent the formation of disulfide bonds and incubated for 30 min at 25°C in the dark. The samples were subsequently diluted with 0.1 M ammonium bicarbonate to have a final concentration of urea of 1.6 M. For digestion, the proteins were incubated overnight at 37°C in presence of 1 μg of trypsin. After acidification with trifluoroacetic acid (TFA, 1% final), the peptides were loaded on a C-18 column (The Nest Group, SS18V) pre-equilibrated with buffer A (0.1% TFA). The column was washed 3 times with buffer C (5% acetonitrile / 95%water (v/v) and 0.1% TFA) and peptides were eluted with buffer B (50% acetonitrile / 50%water (v/v) and 0.1% TFA). The peptides were finally dried under vacuum and kept at—80°C. Before LC-MS/MS mass analysis, samples were resuspended in 0.1% formic acid by sonication.

Mass spectrometry analysis

For each sample, aliquots of 0.4 μg of total peptides were subjected to LC-MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer connected to an electrospray ion source (both Thermo Fisher Scientific) and a custom-made column heater set to 60°C. Peptide separation was carried out using an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75μm × 30cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin; Dr. Maisch GmbH, Germany) using a linear gradient from 95% solvent A (0.1% formic acid in water) and 5% solvent B (80% acetonitrile, 0.1% formic acid, in water) to 35% solvent B over 50 minutes to 50% solvent B over 10 minutes to 95% solvent B over 2 minutes and 95% solvent B over 18 minutes at a flow rate of 0.2 μl/min. The data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 120,000 full width at half maximum (at 400 m/z, MS1) followed by MS/MS (MS2) scans in the linear ion trap of the 20 most intense MS signals. The charged state screening modus was enabled to exclude unassigned and singly charged ions and the dynamic exclusion duration was set to 30 s. The collision energy was set to 35%, and one microscan was acquired for each spectrum.

Protein identification and label-free quantification

The acquired raw-files were imported into the Progenesis QI software (v2.0, Nonlinear Dynamics Limited), which was used to extract peptide precursor ion intensities across all samples applying the default parameters. The generated mgf files were searched using MASCOT against a decoy database containing normal and reverse sequences of the concatenated Homo sapiens (UniProt, Mai 2016) and Bartonella henselae (UniProt, July 2016) proteome and commonly observed contaminants (in total 44102 sequences) generated using the SequenceReverser tool from the MaxQuant software (Version 1.0.13.13). The following search criteria were used: full tryptic specificity was required (cleavage after lysine or arginine residues, unless followed by proline); 3 missed cleavages were allowed; carbamidomethylation (C) was set as fixed modification; oxidation (M) and protein N-terminal acetylation were applied as variable modifications; mass tolerance of 10 ppm (precursor) and 0.6 Da (fragments) was set. The database search results were filtered using the ion score to set the false discovery rate (FDR) to 1% on the peptide and protein level, respectively, based on the number of reverse protein sequence hits in the datasets. Quantitative analysis results from label-free quantification were normalized and statically analyzed using the SafeQuant R package v.2.3.4 (https://github.com/eahrne/SafeQuant/) (PMID: 27345528) to obtain protein relative abundances. This analysis included summation of peak areas per protein and LC MS/MS run followed by calculation of protein abundance ratios. Only isoform specific peptide ion signals were considered for quantification. The summarized protein expression values were used for statistical testing of differentially abundant proteins between conditions. Here, empirical Bayes moderated t-Tests were applied, as implemented in the R/Bioconductor limma package (http://bioconductor.org/packages/release/bioc/html/limma.html). The resulting p-values were adjusted for multiple testing using the Benjamini Hochberg method.

All LC-MS analysis runs are acquired from independent biological samples. To meet additional assumptions (normality and homoscedasticity) underlying the use of linear regression models and Student t-Test MS-intensity signals are transformed from the linear to the log-scale.

Unless stated otherwise linear regression was performed using the ordinary least square (OLS) method as implemented in base package of R v.3.1.2 (http://www.R-project.org/). The sample size of three biological replicates was chosen assuming a within-group MS-signal Coefficient of Variation of 10%. When applying a two-sample, two-sided Student’s t-test this gives adequate power (80%) to detect protein abundance fold changes higher than 1.65, per statistical test. Note that the statistical package used to assess protein abundance changes, SafeQuant, employs a moderated t-Test, which has been shown to provide higher power than the Student’s t-test. We did not do any simulations to assess power, upon correction for multiple testing (Benjamini-Hochberg correction), as a function of different effect sizes and assumed proportions of differentially abundant proteins.

Inhibitor treatment of infected HeLa cells

HeLa cells were seeded at a density of 2’000 cells/well on a 96-well plate (Corning, #3904) using DMEM supplemented with 10% of heat-inactivated FCS. After overnight incubation at 37°C with 5% CO₂, cells were infected with Bhe ΔbepA-G carrying the empty plasmid or expressing BepC at MOI of 400 in M199 (Gibco, 22340–020) supplemented with 10% of heat-inactivated FCS in presence of 10 μM of IPTG. After 24 h of incubation at 35°C and 5% CO₂, the medium was removed and cells were incubated with inhibitor diluted in DMEM at 35°C with 5% CO₂. The treatment consisted of Rho inhibitor I (Cytoskeleton, CT04) for 2 h or Y27632 (Sigma, Y0503) for 1 hour at the indicated concentration. The experiment was stopped by fixation with 3.7% of paraformaldehyde for 10 minutes. Finally, the cells were washed 3 times with PBS before being stained and imaged by microscopy.

Generation of knock-out cells

HeLa cells were seeded at a density of 1’400’000 cells per 150 cm2 flask in DMEM supplemented with 10% of heat-inactivated FCS and incubated overnight at 37°C with 5% CO₂. The day after, in 1.2 ml of DMEM without FCS, 12 μg of plasmid encoding GFP and the guide RNA (gRNA) targeting the first exon of the gene of interest were mixed with 12 μg of plasmid carrying the puromycin resistance gene and the gRNA targeting the last exon. After adding 48 μl of FuGene HD transfection reagent (Promega, E2311), the transfection mix was incubated for 15 min at room temperature before being transferred in the cell culture flask. The cells were transfected for 24 h at 37°C with 5% CO₂. Double-transfected cells were selected in the presence of puromycin (1.5 μg/ml) for 24 h at 37°C with 5% CO₂ followed by FACS to select GFP-positive cells. Selected cells were collected in DMEM with 10% heat-inactivated FCS supplemented with penicillin-streptomycin and expanded for several days. Finally, cells were stored at -80°C in DMEM supplemented with 10% heat-inactivated FCS and 10% DMSO. The expression level of the protein of interest was monitored via immunoblot.

Immunoblot analysis

The samples used for immunoblot analysis were separated by SDS-PAGE on a 4–20% gradient gel (Mini-PROTEAN TGX Gels, Biorad, Cat# 456–1093). Gel electrophoresis was performed at 120 V in running buffer (Tris-glycine, 0.1% SDS). Proteins were transferred on a PVDF membrane (GE Healthcare, 10600021) via wet electroblotting at 100 V in transfer buffer (20% methanol, Tris-glycine) at 4°C. After transfer, the membrane was incubated for 1 hour in blocking buffer (PBS, 0.1% Tween 20 (Sigma, 93773), supplemented with 5% milk or 5% BSA according to antibody recommendation). After washing with PBS 0.1% Tween 20, the membrane was incubated overnight at 4°C in blocking buffer with the primary antibody (S8 Table). The membrane was washed again with PBS 0.1% Tween 20 before being incubated 1 hour at room temperature in blocking buffer with the secondary antibody (S8 Table). The blots were developed using LumiGLO Reserve Chemiluminescent Substrate System (KPL, 54-70-00, 54-69-00). Finally, the signal was detected with LAS4000 (Fujifilm).

Quantification of F-actin and pMLC via Cellprofiler

Experiments performed in 96-well plates were subjected to automated microscopy, using MD ImageXpress Micro automated microscopes. For each condition, at least 6 wells with 25 sites were imaged in 4 different wavelengths corresponding to the applied cell staining (DAPI, DY-547P1 phalloidin, pMLC, Bartonella). Images were analyzed with the CellProfiler software [41]. Two separate Cellprofiler pipelines are used for each assay. The first pipeline calculates a shading model, which is used by the second pipeline to correct images prior to analysis. To correct uneven illumination inherent in wide-field microscopic imaging (shading), an illumination function was computed. The illumination function was calculated on all images based on the Background method. The resulting image was smoothed using a Gaussian method with a 100-filter size. To reduce the signal originating from the bacterial DNA in the DAPI channel, the signal corresponding to Bartonella was subtracted from the DAPI image. On all images, CellProfiler was executed to perform object segmentation and measurements with the following steps. Nuclei were detected as primary objects using an Automatic strategy and clumped objects were identified based on their shape and segmented based on their intensity. HeLa cells were detected as secondary objects via their DY-547P1 phalloidin signal by using a Propagation method from the nuclei followed by a Global threshold strategy combined with an Otsu threshold method. The average of the mean intensity of each cell within one site was measured for the F-actin or pMLC signals. Data from all sites from the same conditions were compiled together. The mean cell intensity per site was normalized on the uninfected condition.

Statistical analysis

Graph was generated with GraphPad Prism 8. When data were not following a normal Gaussian distribution, statistical analyses were performed using Kruskal-Wallis test with Dunn’s multiple comparison test. For the graph presented in the figures, significance was denoted as ajusted P-value P* < 0.1, P** < 0.01, P*** < 0.001, P**** < 0.0001.

Software

ImageJ [42] was used to create z-projection and x-z sections of confocal microscopy images. MetaXpress (Molecular Devices) was used to acquire and generate microscopy pictures from automated microscope. Image analysis and the calculation of the average of the mean cell fluorescence intensity was realized via Cellprofiler [41]. GraphPad prism 8 (GraphPad) was used to create the dot-plot and the statistical analysis. Geneious Prime 2019 (Geneious) was used to design cloning of plasmids. The schematic model of BepC-mediated actin stress fiber formation was created with Biorender.com.