Targeting the BspC-vimentin interaction to develop anti-virulence therapies during Group B streptococcal meningitis
Fig 5
The V-domain pocket contributes to BspC Function (A) Zoomed in view of the V-domain pocket with residues targeted for mutagenesis. Residues shown in blue were targeted to construct LPM, and residues shown in green were targeted to construct RPM. Visualization done using PyMOL. (B) COH1 ΔbspC GBS containing pDCErmbspC (pbspC), pDCErmbspC LPM (pLPM), pDCErmbspC RPM (pRPM) or the empty vector (pDCErm) were stained with an anti-BspC antibody or isotype control without permeabilization. Surface bound antibody was measured via flow cytometry. (C) The same strains used in (B) were used to infect a monolayer of hCMECs. CFU were plated to assess adherence after 30 minutes of incubation. (D-H) Mice were infected with ~4x108 CFU of the same strains used in (B) and then sacrificed after 48 hours. (D) Half of each mouse brain was homogenized and plated on THA-Erm to determine bacterial burden. (E) Blood from each mouse was also plated on THA-Erm to determine bacterial burden. For (D) and (E), data is pooled from two independent experiments, each dot represents an individual mouse, and lines indicate statistical means. (F) H&E stains showing the leptomeninges from two individual mice per group, one from each independent experiment. Arrows indicate areas of leukocyte infiltration and meningeal thickening. (G) ImageJ was used to analyze meningeal thickness from 5 mice per group from the two independent experiments. Two images were taken of each brain, and three points per image were measured with each dot representing an individual point. (H) KC protein concentration was quantified via ELISA from brain homogenates shown in D. Data for H is pooled from two independent experiments, where each dot represents an individual mouse brain, and lines indicate statistical means. Statistical analysis: (C, D, E, G, and H) One-way ANOVA with Tukey’s multiple comparisons. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005.