Bacterial induction of Snail1 contributes to blood-brain barrier disruption

Abstract

Bacterial meningitis is a serious infection of the CNS that results when blood-borne bacteria are able to cross the blood-brain barrier (BBB). Group B Streptococcus (GBS) is the leading cause of neonatal meningitis; however, the molecular mechanisms that regulate bacterial BBB disruption and penetration are not well understood. Here, we found that infection of human brain microvascular endothelial cells (hBMECs) with GBS and other meningeal pathogens results in the induction of host transcriptional repressor Snail1, which impedes expression of tight junction genes. Moreover, GBS infection also induced Snail1 expression in murine and zebrafish models. Tight junction components ZO-1, claudin 5, and occludin were decreased at both the transcript and protein levels in hBMECs following GBS infection, and this repression was dependent on Snail1 induction. Bacteria-independent Snail1 expression was sufficient to facilitate tight junction disruption, promoting BBB permeability to allow bacterial passage. GBS induction of Snail1 expression was dependent on the ERK1/2/MAPK signaling cascade and bacterial cell wall components. Finally, overexpression of a dominant-negative Snail1 homolog in zebrafish elevated transcription of tight junction protein-encoding genes and increased zebrafish survival in response to GBS challenge. Taken together, our data support a Snail1-dependent mechanism of BBB disruption and penetration by meningeal pathogens.

Figure 8. Inhibition of Snail1 impacts expression of junctional components and reduces GBS virulence in zebrafish larvae.

Transgenic zebrafish expressing an inducible dominant-negative snai1a, Tg(HSP:dn-snai1a), upregulated occludin, E-cadherin, and claudin 5 following heat shock (HS) (n = 9) when compared with controls with no heat shock (no HS) (n = 4) 6 hours after heat shock (A–C). Kaplan-Meier survival curve of WT or Tg(HSP:dn-snai1a) transgenic zebrafish following heat shock and GBS challenge (n = at least 10/group). Transgenic zebrafish exhibited significantly lower mortality rates following GBS challenge compared with those of WT zebrafish (D). Data represent 3 independent experiments combined. Student’s t and log-rank tests were used to determine significance. *P < 0.05; **P < 0.01.

Figure 7. Contribution of Snail1 to GBS-BBB penetration in vivo.

Kaplan-Meier survival curve for zebrafish (n = 9/group) infected with GBS (A). Representative images of GBS-infected zebrafish showing cerebral swelling and edema compared with noninfected controls (B). GBS infection increased snai1a transcript levels in brain homogenates of zebrafish following GBS infection (n = 19) compared with those in noninfected zebrafish (n = 12) (C). Knockdown of snai1a using siRNA duplexes (n = 10/treatment group) did not change GBS bloodstream survival, but attenuated bacterial penetration into the brain (D). Representative data from 1 of 2 independent experiments are shown. Student’s t and log-rank tests were used to determine significance. *P < 0.05; ***P < 0.001.

Figure 6. Contribution of MAPK signaling in SNAI1 expression.

hBMECs were treated with either DMSO vehicle control or a specific MAPK pathway inhibitor. U0126 had the greatest effect on inhibition of GBS-induced SNAI1 expression (A). Treatment of hBMECs with the TLR2 agonist P3C resulted in a dose-dependent increase in SNAI1 expression (B). Treatment of hBMECs with extracts from Δiag or HY106 Δiag resulted in significantly lower SNAI1 expression levels (C). Experiments were performed at least 3 times in triplicate; error bars represent the SEM of at least 3 biological replicates. One-way ANOVA was used to determine significance. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. GBS factors contribute to SNAI1 expression.

Fixed GBS was able to induce SNAI1 expression at levels that were comparable to those in live GBS; however, HK GBS was not able to induce SNAI1 (A). Cell wall extracts (CWE) prepared from WT GBS were greatly able to induce SNAI1 (B). Experiments were performed at least 3 times in triplicate; error bars represent the SEM of at least 3 biological replicates. One-way ANOVA was used to determine significance. **P < 0.01; ***P < 0.001.

Figure 4. Snail1 is sufficient to disrupt brain endothelial tight junctions.

Overexpression of Snail1 resulted in a loss of tight junction proteins (A–D). Control cell line shows the expected cobblestone pattern, and overexpression of Snail1 resulted in a loss of tight junction staining (scale bars: 20 μm) (E). Snail1 overexpression increased hBMEC permeability as assessed by Evans Blue dye migration after induction with doxycycline; dye was added to the upper chamber and quantified colorimetrically in the lower chamber (OD600) (F). Experiments were performed at least 3 times in triplicate; error bars represent the SEM of at least 3 biological replicates (F), or the SD for protein analysis of a representative experiment (B–D). Student’s t test was used to determine significance. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Snail1 is necessary to disrupt brain endothelial tight junctions.

shRNA knockdown of Snail1 in hBMECs resulted in a significant reduction of Snail1 protein abundance (A and B). SNAI1 expression was no longer upregulated upon GBS infection with shRNA knockdown (C). Occludin transcript and protein levels, with knockdown of SNAI1 during GBS infection (D–F). shRNA hBMEC barrier function was greater as visualized by Evans Blue migration; dye was added to the upper chamber and quantified colorimetrically in the lower chamber (OD600) (G). Experiments were performed at least 3 times in triplicate; error bars represent the SEM of at least 3 biological replicates (C, D, and G), or the SD of a representative experiment (B and F). One-way ANOVA (C, D, and F) and Student’s t test (B and G) were used to analyze statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. GBS disrupts tight junctions of brain endothelium.

GBS decreased transcript abundance of zo-1 (A), occludin (B), and claudin 5 (C) in confluent hBMECs (A and B) or bEND3 (C) following GBS infection for 5 hours at an MOI of 10. qPCR experiments were performed at least 3 times in triplicate. Error bars represent the SEM of at least 3 biological replicates (A–C). Protein levels of ZO-1 and occludin in hBMECs (D, F, and G) or bEND3 (E and H) during GBS infection were determined by Western blot analysis. Lysates were probed for ZO-1, occludin, or claudin 5. Western blot analysis was normalized to GAPDH (D–H). Experiments were performed at least 3 times in triplicate. Error bars represent the SD of a representative experiment (F–H). hBMECs were stained for ZO-1 and visualized by immunofluorescence (scale bars: 20 μm) (I). Mice (n = 8/group) were infected i.v. with GBS or vehicle control. Upon sacrifice, brain tissue was collected and endothelial cells isolated for RNA extraction and qPCR analysis (J and K). Student’s t test was used for image analysis. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 1. GBS upregulates Snail1 in brain endothelium.

Upregulation of SNAI1 transcription as assessed by microarray analysis during infection with GBS, SPN, HiB, and B.a. ΔpX01 (B.aΔ) (A). Changes in mRNA transcripts in confluent hBMECs or bEND3 monolayers after a 5-hour infection with GBS at an MOI of 10) (B and C). Confluent hBMECs were infected for 5 hours with different serotypes of GBS at an MOI of 10 (type Ia and III) or an MOI of 0.1 (type V). RNA was extracted and SNAI1 transcripts analyzed (D). Changes in protein levels of confluent hBMEC monolayers after infection for 5 hours with GBS at an MOI of 10 (E and F). Mice (n = 8/group) were infected i.v. with GBS or vehicle control. Upon sacrifice, brain tissue was collected and endothelial cells isolated for RNA extraction and qPCR analysis (G). Brains of infected mice (n = 10) showed Snail1 colocalized with VWF factor (scale bar: 50 μm) (H and I). Experiments were performed at least 3 times in triplicate. For qPCR analysis, error bars represent the SEM of at least 3 biological replicates (B–D and G), or the SD for protein analysis of a representative experiment (F). Student’s t test was used to determine statistical significance. *P < 0.05; **P < 0.01.