Fig 1.
BifA enhances SEZ virulence, brain colonization and disrupts the BBB.
(A) Recovery of WT or ΔBif CFU from the indicated organs of BALB/c mice 48 hr after i.p. challenge with 1×105 CFU of either SEZ strain (**** indicates p-value <0.0001 with t test). (B) Survival curves of BALB/c mice after i.p. challenge with 5×105 CFU WT, ΔBif, or ΔBif complemented with WT BifA (CBif), or BifA deleted for the Fic domain (CBif (ΔFic)), or a BifA H247A substitution mutant (CBif (H247A)). Each group contains 20 mice (**** indicates p value <0.0001 with Log-rank Test vs WT). (C) Burdens of indicated strains in mouse brains 2 days post i.p. challenge with 5×105 CFU, 10 mice/group (**** indicates p-value <0.0001 with t test). (D) Evans Blue (EB) permeability in brains of mice infected with the indicated strains. Mice were inoculated with EB 18 hours after they were i.v. inoculated with 5×106 CFU of WT, ΔBif or CBif. Six hours later, brains were dissected and EB was extracted and quantified (** indicates p-value <0.01 and ***indicates p-value <0.001with t test). (E) Ratio of bacterial burdens in cerebrospinal fluid versus blood 12h post i.v. infection with indicated strains (4 mice/group, **** indicates p-value <0.0001 with t test). (F) WT and mutant SEZ traversal of hBMEC grown in transwell chambers. The Y-axis represents the percentage of bacteria that migrated from the upper to the lower chamber in 3 replicates (**** indicates p-value <0.0001 with t test).
Fig 2.
BifA is released into culture supernatants and enters into host cells.
(A) Detection of BifA in culture supernatants by western blot. Immunoblots were performed with anti-BifA polyclonal antibody. Immunoblots of supernatants with antibody against GroEL, a cytosolic protein, were also performed to check for cytosolic contamination of supernatants and of bacterial lysates as a loading control. (B) Immunofluorescence microscopy of hBMECs incubated with supernatants from WT or ΔBif SEZ. BifA was detected by immunostaining with anti-BifA antibody (green) and DAPI was used as a counterstain for nuclei (blue) (scale bar = 10 μm). (C) Diagram of wild type and variant BifA proteins purified for experiments in (D) and (E). (D) Immunofluorescence microscopy of hBMECs that had been incubated with indicated BifA variants. BifA was detected by immunostaining (green) and DAPI was used as a counterstain for nuclei (blue). Images represent merged fluorescence channels (scale bar = 50 μm). (E) Transmission EM of hBMECs that had been incubated with latex beads coated with indicated BifA variants. For mock, cells were incubated with uncoated latex beads. Dashed boxes indicate areas of higher magnification (shown on right for BifA and ΔRhuM). Arrows indicate internalized latex beads (scale bar = 1 μm).
Fig 3.
BifA disrupts hBMEC monolayer barrier function.
(A) Transwell-grown hBMECs were treated with purified BifA or its variants and assessed for barrier integrity with an Evans Blue dye penetration assay (quantitated by absorbance at OD600) (results are from 3 experiments, * indicates p-value <0.05, ** indicates p-value <0.01, *** indicates p-value <0.001 with t test). (B) Time-lapse microscopy of hBMEC monolayers following addition of purified BifA, BifA H247A, or mock treatment with DMEM. Scale bar = 50 μm. For the movies, see supplemental video (S1–S6 Movies). (C) Quantitation of gap size in S2 Movie (using Image J) detected in monolayers 2 and 3 hours after treatment with BifA. (D) Immunoblot detection of ZO-1 in lysates of hBMEC after BifA treatment. GAPDH was used as reference protein.
Fig 4.
(A) Schematic of proteomic strategy for identifying BifA binding partner(s). 293T cells were transfected with plasmids encoding GFP-tagged BifA or GFP alone, differentially labelled with heavy isotope (Arg 13C6, Lys 13C6) or light isotope (Arg 12C6, Lys 12C6), and GFP or GFP-BifA binding partners were compared by SILAC (see methods for details). (B) Co-immunoprecipitation of HA-tagged and GFP tagged BifA or its variants. Inputs are immunoblots of moesin and BifA in cell lysates prior to co-IP. Outputs are immunoblots of moesin and BifA in eluates after BifA immunoprecipitation. (C) Schematic of moesin variants for experiments in D and E. (D) Co-immunoprecipitation of GFP-tagged BifA and HA-tagged moesin variants, immunoblotted as in (B). (E) Surface plasmon resonance of the moesin-BifA interaction. The Y-axis shows response units (RU), where 1 RU is equivalent to a change in surface protein concentration of 1 pg/mm2.
Fig 5.
BifA leads to moesin T558 phosphorylation.
(A-D) Western blot detection of moesin phosphorylation upon BifA treatment. Human BMEC cells were incubated with different BifA variants for the indicated times and lysates were probed with appropriate antibody. GAPDH is a loading control. The graphs on the right panel show the quantitation of the moesin and phospho-moesin bands by ImageJ, normalized with GAPDH. (E) Effect of PKC inhibition upon BifA induction of moesin phosphorylation. Cells were pre-treated with PKC inhibitor NSC305787 for 30 min before BifA treatment, then analyzed by immunoblot with phospho-moesin antibody. (F) BifA treatment leads to formation of GTP-RhoA. Western blots were performed on lysates of cells treated with BifA alone, or BifA in the presence of NSC305787. Total RhoA and rhotekin protein precipitated GTP-RhoA was detected by anti-RhoA antibody. GAPDH was the loading control.
Fig 6.
Inhibition of moesin phosphorylation or moesin knockdown block BifA disruption of monolayer barrier functions.
(A) NSC305787 treatment and moesin KD prevent BifA-induced increase in monolayer permeability. Transwell-grown hBMEC monolayers were assayed for barrier integrity by Evans Blue dye (data represent 3 replicates /condition, *** indicates p-value <0.001 with t test). (B) NSC305787 treatment or moesin KD inhibit SEZ penetration of hBMEC monolayers. Transwell-grown hBMEC monolayers were infected with SEZ by inoculation into the upper chamber, and the proportion of bacteria in the lower chamber were quantitated by CFU plating (data represent 3 replicates /condition, **** indicates p-value <0.0001 with t test).