Bacterial induction of Snail1 contributes to blood-brain barrier disruption
Open accessAbstract
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.
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Written from the paper’s full text.
TL;DR
GBS and other meningeal pathogens induce the host transcriptional repressor Snail1 in brain endothelium, which represses tight junction genes and promotes blood-brain barrier disruption. Snail1 is necessary and sufficient for this loss of barrier integrity, and inhibiting Snail1 reduces GBS brain penetration and improves survival in zebrafish.
Key findings
- Infection of hBMECs with GBS, SPN, and B.a.ΔpXO1, but not HiB, significantly induced SNAI1 by microarray; qPCR confirmed SNAI1 induction in hBMECs and bEND3 cells and Snail1 protein induction with GBS, and GBS-infected mice showed significantly increased SNAI1 transcripts in isolated brain endothelial cells.
- GBS infection decreased ZO-1, occludin, and claudin 5 at transcript and protein levels in hBMECs/bEND3 and in mouse brain endothelial cells; Snail1 knockdown rescued occludin and reduced Evans Blue migration, while Snail1 overexpression decreased ZO-1, occludin, and VE-cadherin and increased Evans Blue permeability.
- GBS induction of SNAI1 was dependent on MEK1/2/ERK1/2 signaling (U0126 reduced SNAI1), was induced by formalin-fixed but not heat-killed GBS, was induced by GBS cell wall extract, and was reduced with Δiag and HY106/Δiag LTA-anchoring mutants; the TLR2 agonist P3C dose-dependently increased SNAI1.
- In zebrafish, GBS caused up to 90% mortality and cerebral swelling/edema; siRNA knockdown of snai1a reduced GBS recovered from brain but not blood, and heat-shocked Tg(HSP:dn-snai1a) larvae had increased occludin, claudin 5, and E-cadherin transcripts and about 40% survival versus 10% of WT larvae at 72 hours after infection.
Methods
- Comparative microarray analysis of hBMECs infected with GBS, SPN, HiB, and B. anthracis, followed by qPCR, Western blot, and immunofluorescence; in vitro infections used GBS at MOI 10 for 4–5 hours, MAPK inhibitors U0126, SB202190, and SP600125 at 20 μM, TLR2 agonist P3C, and GBS cell wall extracts.
- Lentiviral doxycycline-inducible shSNAI1 knockdown and Snail1-overexpression hBMEC lines; Transwell Evans Blue permeability assays; murine hematogenous meningitis model using 6- to 8-week-old male CD-1 mice injected i.v. with 6 × 10⁷ to 7 × 10⁷ CFU WT GBS or vehicle, with brain endothelial cell isolation.
- Adult and larval zebrafish GBS infection models, siRNA snai1a knockdown, Tg(HSP:dn-snai1a) transgenic zebrafish, bacterial enumeration, qPCR, and survival analysis; statistics used 2-tailed Student’s t test, 1-way ANOVA with Bonferroni’s post test, and log-rank tests, with significance at P < 0.05.
Limitations
- Stated: Snail1-KO mice are embryonically lethal, so the in vivo functional role of Snail1 was examined in zebrafish; future studies are needed to determine the mechanism of LTA activation and whether other LTA-associated proteins play a role.
- Stated: no increase in SNAI1 transcript levels was observed in hBMECs upon addition of TNF-α or IL-1β (data not shown), and some supporting data are not shown.
- Evident from the text: the findings rely on immortalized hBMEC/bEND3 cell lines and murine/zebrafish models, with no human clinical samples or direct human in vivo validation reported.
Model-written summary. Check it against the paper before relying on it.
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- Findingown data
Infection of hBMECs with GBS and other meningeal pathogens (SPN, B.a.) but not HiB induces SNAI1 transcription.
“Data analysis of these experiments using a statistical algorithm developed for high-density oligonucleotide arrays (25) revealed that infection with all pathogens, with the exception of HiB, resulted in significant induction of SNAI1 (Figure 1A).”
- Also stated in: § Introduction · plain
“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.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Also stated in: § Introduction · plain
- Findingown data
SNAI1 knockdown reduces Evans Blue migration across hBMEC monolayers in response to GBS infection.
“In response to GBS infection, less Evans Blue migration was observed in doxycycline-treated shSNAI1-hBMECs (Figure 3G).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingown data
GBS infection increases Snail1 protein levels in hBMECs.
“We further analyzed protein expression by Western blot analysis and found that Snail1 protein levels were significantly induced during GBS infection (Figure 1, E and F).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingcitation
Inhibition of MEK1/2/ERK1/2 with U0126 reduces GBS-induced SNAI1 transcripts.
“Inhibition of the MEK1/2/ ERK1/2 components of the MAPK pathway with U0126 resulted in a significant reduction of SNAI1 transcripts in response to GBS as described previously (23,27).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingown data
Inhibitors of p38 and JNK have no effect on GBS-induced SNAI1.
“while inhibitors of p38 (SB202190) and JNK (SP600125) had no effect (Figure 6A).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingown data
GBS infection increases SNAI1 transcripts in brain endothelial cells of mice in vivo.
“SNAI1 transcripts were significantly increased in GBS-infected mice compared with levels in control mice (Figure 1G).”
- Also stated in: § Discussion · plain
“Using a murine model of hematogenous meningitis, we observed a significant increase in SNAI1 transcripts in brain tissue during GBS infection when compared with noninfected controls.”
Quote in the paperSubstantial claim - Also stated in: § Discussion · plain
- Findingown data
GBS does not induce SNAI1 in doxycycline-treated shSNAI1-hBMECs.
“As expected, GBS did not induce SNAI1 in doxycycline-treated shSNAI1-hBMECs (Figure 3C).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingown data
Snail1 overexpression increases Evans Blue permeability in hBMEC-TRE:SNAI1.
“Increased permeability to Evans Blue was observed in doxycycline-treated hBMEC-TRE:SNAI1 when compared with that in untreated cells (Figure 4F).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingown data
TLR2 agonist P3C induces SNAI1 in a dose-dependent manner in hBMECs.
“We observed a dose-dependent increase in SNAI1 in response to P3C (Figure 6B).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingown data
GBS cell wall extract induces SNAI1 transcript levels in hBMECs.
“Strikingly, GBS cell wall extract greatly induced SNAI1 transcript levels when compared with those of the control (Figure 5B).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Findingown data
GBS infection reduces claudin 5 and occludin expression in brain endothelial cells isolated from infected mice.
“Finally, endothelial cells isolated from infected mice had significantly lower claudin 5 and occludin expression levels when compared with those in uninfected mice (Figure 2, J and K).”
Quote in the paperSubstantial claim - Findingown data
Snail1 colocalizes with VWF in brain tissue during GBS infection, indicating expression in endothelial cells.
“We further examined the localization of Snail1 in brain tissue and observed that Snail1 colocalized with von Willebrand factor (VWF), further supporting the observation that Snail1 is expressed in endothelial cells during active infection (Figure 1, H and I).”
Quote in the paperSubstantial claim - Findingown data
GBS infection upregulates snai1a in brain tissue of adult zebrafish.
“As shown in Figure 7C, GBS significantly upregulated snai1a in the brain tissue of infected zebrafish.”
Quote in the paperSubstantial claim - Findingown data
In brains of GBS-infected mice, Snail1 colocalizes with VWF factor.
“Brains of infected mice (n = 10) showed Snail1 colocalized with VWF factor (scale bar: 50 μm) (H and I).”
Quote in the paperSubstantial claim - Findingown data
Heat-shocked HSP:dn-snai1a zebrafish larvae have increased transcript levels of occludin, claudin 5, and E-cadherin.
“Heat-shocked HSP:dn-snai1a larvae 3 days post fertilization (dpf) had increased transcript levels of occludin, claudin 5, and E-cadherin compared with those in larvae that were not heat shocked (Figure 8, A-C).”
Quote in the paperSubstantial claim- Rests on an assumption: The Tg(HSP:dn-snai1a) zebrafish line functions as a dominant-negative inhibitor of endogenous snai1a.
- Findingown data
siRNA knockdown of snai1a decreases GBS recovered from brain tissue but not blood in infected adult zebrafish.
“Injection of siRNA decreased brain snai1a transcripts (Supplemental Figure 3E) and resulted in a significant decrease in the amount of GBS recovered from brain tissue of infected fish (Figure 7D), while no differences in bacterial levels recovered from the blood were observed (Supplemental Figure 3F).”
Quote in the paperSubstantial claim - Findingown data
SNAI1 transcription is upregulated during infection with GBS, SPN, HiB, and B.a. ΔpX01 (B.aΔ) as assessed by microarray analysis.
“Upregulation of SNAI1 transcription as assessed by microarray analysis during infection with GBS, SPN, HiB, and B.a. ΔpX01 (B.aΔ) (A).”
Quote in the paperSubstantial claim - Findingown data
GBS upregulates Snail1 in brain endothelium.
“GBS upregulates Snail1 in brain endothelium.”
Quote in the paperSubstantial claim - Findingown data
HSP:dn-snai1a larvae have higher survival after GBS challenge than WT larvae (about 40% vs 10% at 72 h).
“At 72 hours after infection, only 10% of WT larvae survived, whereas about 40% of the HSP:dn-snai1a larvae were still viable (Figure 8D).”
Quote in the paperSubstantial claim- Rests on an assumption: The Tg(HSP:dn-snai1a) zebrafish line functions as a dominant-negative inhibitor of endogenous snai1a.
- Findingown data
Transgenic zebrafish expressing inducible dominant-negative snai1a (Tg(HSP:dn-snai1a)) upregulate occludin following heat shock compared with controls with no heat shock, 6 hours after heat shock.
“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).”
Quote in the paperSubstantial claim - Findingown data
GBS infection for 5 hours at an MOI of 10 decreases occludin transcript abundance in confluent hBMECs.
“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.”
Quote in the paperSubstantial claim - Findingown data
Knockdown of snai1a using siRNA duplexes attenuates bacterial penetration into the brain.
“Knockdown of snai1a using siRNA duplexes (n = 10/treatment group) did not change GBS bloodstream survival, but attenuated bacterial penetration into the brain (D).”
Quote in the paperSubstantial claim - Findingown data
GBS infection for 5 hours at an MOI of 10 decreases claudin 5 transcript abundance in confluent bEND3.
“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.”
Quote in the paperSubstantial claim - Findingown data
Transgenic zebrafish expressing inducible dominant-negative snai1a (Tg(HSP:dn-snai1a)) upregulate claudin 5 following heat shock compared with controls with no heat shock, 6 hours after heat shock.
“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).”
Quote in the paperSubstantial claim - Findingown data
Snail1 overexpression increases hBMEC permeability as assessed by Evans Blue dye migration after doxycycline induction.
“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).”
Quote in the paperSubstantial claim - Findingown data
Treatment of hBMECs with extracts from Δiag or HY106 Δiag results in significantly lower SNAI1 expression levels.
“Treatment of hBMECs with extracts from Δiag or HY106 Δiag resulted in significantly lower SNAI1 expression levels (C).”
Quote in the paperSubstantial claim - Findingown data
GBS infection increases snai1a transcript levels in brain homogenates of zebrafish compared with noninfected zebrafish.
“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).”
Quote in the paperSubstantial claim - Findingown data
U0126 has the greatest effect on inhibition of GBS-induced SNAI1 expression among the MAPK pathway inhibitors tested.
“U0126 had the greatest effect on inhibition of GBS-induced SNAI1 expression (A).”
Quote in the paperSubstantial claim - Findingown data
Cell wall extracts prepared from WT GBS greatly induce SNAI1.
“Cell wall extracts (CWE) prepared from WT GBS were greatly able to induce SNAI1 (B).”
Quote in the paperSubstantial claim - Findingown data
GBS-infected zebrafish show cerebral swelling and edema compared with noninfected controls.
“Representative images of GBS-infected zebrafish showing cerebral swelling and edema compared with noninfected controls (B).”
Quote in the paperSubstantial claim - Findingown data
hBMECs with shRNA knockdown of Snail1 have greater barrier function as assessed by Evans Blue migration.
“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).”
Quote in the paperSubstantial claim - Findingown data
Overexpression of Snail1 results in a loss of tight junction proteins.
“Overexpression of Snail1 resulted in a loss of tight junction proteins (A-D).”
Quote in the paperSubstantial claim - Findingown data
Treatment of hBMECs with the TLR2 agonist P3C results in a dose-dependent increase in SNAI1 expression.
“Treatment of hBMECs with the TLR2 agonist P3C resulted in a dose-dependent increase in SNAI1 expression (B).”
Quote in the paperSubstantial claim - Findingown data
Transgenic zebrafish expressing inducible dominant-negative snai1a (Tg(HSP:dn-snai1a)) upregulate E-cadherin following heat shock compared with controls with no heat shock, 6 hours after heat shock.
“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).”
Quote in the paperSubstantial claim - Findingown data
Knockdown of snai1a using siRNA duplexes does not change GBS bloodstream survival.
“Knockdown of snai1a using siRNA duplexes (n = 10/treatment group) did not change GBS bloodstream survival, but attenuated bacterial penetration into the brain (D).”
Quote in the paperSubstantial claim - Findingown data
With shRNA knockdown of Snail1, SNAI1 expression is no longer upregulated upon GBS infection.
“SNAI1 expression was no longer upregulated upon GBS infection with shRNA knockdown (C).”
Quote in the paperSubstantial claim - Findingown data
Fixed GBS induces SNAI1 expression at levels comparable to live GBS.
“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).”
Quote in the paperSubstantial claim - Findingown data
GBS infection for 5 hours at an MOI of 10 decreases zo-1 transcript abundance in confluent hBMECs.
“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.”
Quote in the paperSubstantial claim - Findingown data
shRNA knockdown of Snail1 in hBMECs significantly reduces Snail1 protein abundance.
“shRNA knockdown of Snail1 in hBMECs resulted in a significant reduction of Snail1 protein abundance (A and B).”
Quote in the paperSubstantial claim - Findingown data
Transgenic zebrafish expressing inducible dominant-negative snai1a exhibit significantly lower mortality rates following GBS challenge compared with WT zebrafish.
“Transgenic zebrafish exhibited significantly lower mortality rates following GBS challenge compared with those of WT zebrafish (D).”
Quote in the paperSubstantial claim - Findingown data
Heat-killed GBS does not induce SNAI1.
“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).”
Quote in the paperSubstantial claim - Findingown data
Overexpression of Snail1 results in a loss of tight junction staining.
“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).”
Quote in the paperSubstantial claim - Interpretationown data
Snail1 expression is necessary for GBS-mediated downregulation and disruption of tight junction proteins and decreased endothelial barrier integrity.
“These results suggest that Snail1 expression is necessary for GBS-mediated downregulation and disruption of tight junction proteins, which results in decreased endothelial barrier integrity.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Study design and conductown data
Doxycycline-induced Snail1 overexpression in hBMEC-SNAI1 increases Snail1 protein.
“When treated with doxycycline, protein levels of Snail1 were increased in hBMEC-SNAI1 compared with levels in the noninduced controls (Figure 4, A and B).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Study design and conductown data
Doxycycline-induced shSNAI1 reduces Snail1 in the hBMEC line.
“The generated hBMEC line (shSNAI1-hBMEC) was treated with doxycycline to induce expression of shSNAI1, which resulted in a reduction of Snail1 (Figure 3, A and B).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Interpretationown data
Snail1 expression is sufficient to cause loss of tight junctions and barrier integrity in brain endothelium.
“These data suggest that Snail1 expression is sufficient to cause the loss of tight junctions and subsequent barrier integrity in brain endothelium.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Interpretationown data
GBS LTA may induce SNAI1 expression.
“Our results also suggest that GBS LTA may induce SNAI1 expression.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Premisecitation
Snail1-KO mice are embryonically lethal.
“As the Snail1-KO mice are embryonically lethal (31), we developed an in vivo model of GBS infection using Danio rerio (zebrafish) adults.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [31] source
Not checked in the free run.
- Interpretationown data
Inhibition of Snail1a is sufficient to protect zebrafish larvae from GBS infection.
“This animal showed increased survival when compared with WT animals, suggesting that inhibition of Snail1a is sufficient to protect zebrafish larvae from GBS infection.”
Quote in the paperSubstantial claim- Rests on an assumption: The Tg(HSP:dn-snai1a) zebrafish line functions as a dominant-negative inhibitor of endogenous snai1a.
- Premisecitation
SNAI1-null alleles are embryonically lethal at E8.5.
“Further experiments with murine models are technically challenging, as SNAI1-null alleles are embryonically lethal at E8.5 (31,56,57).”
Quote in the paperSubstantial claim0 of 3 sources checked - Interpretationown data
Snail1-dependent mechanism of BBB disruption and penetration by meningeal pathogens.
“Taken together, our data support a Snail1-dependent mechanism of BBB disruption and penetration by meningeal pathogens.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Premisecitation
Host ECM components and receptors preferentially localize to basolateral surface of polarized BBB endothelium.
“Given that host ECM components and receptors preferentially localize to the basolateral surface of polarized BBB endothelium (16), we hypothesized that disruption of junctional protein complexes in brain endothelium is the first step leading to bacterial”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [16] source
Not checked in the free run.
- Premisecitation
Snail1 binds promoter regions of occludin, claudin family, and other adherence junction protein genes.
“Snail1 is a global repressor of tight junctions, as Snail1 binds the promoter regions of the gene encoding occludin, genes in the claudin family, and genes of other adherence junction proteins (21,22).”
Quote in the paperSubstantial claim0 of 2 sources checked - Interpretationown data
GBS disrupts tight junctions of brain endothelium.
“GBS disrupts tight junctions of brain endothelium.”
Quote in the paperSubstantial claim - Interpretationreasoning
Loss of tight junctions may represent critical first step enabling bacterial pathogens to engage basolaterally expressed host receptors and promote BBB disruption and progression to meningitis.
“The subsequent loss of tight junctions may represent the critical first step that enables bacterial pathogens to engage basolaterally expressed host receptors and promote BBB disruption and progression to meningitis.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Premisecitation
Δiag mutant lacks ability to properly anchor LTA.
“We have previously described a GBS mutant (Δiag) that lacks the ability to properly anchor (LTA) (6), a known TLR2 ligand (30).”
Quote in the paperSubstantial claim0 of 2 sources checked - Interpretationown data
This is first characterization of role of Snail1 in pathogenesis of bacterial infection in vivo.
“To our knowledge, this is the first characterization of the role of Snail1 in the pathogenesis of bacterial infection in vivo.”
Quote in the paperSubstantial claim - Study design and conductown data
Heat shocking WT larvae does not increase occludin, claudin 5, or E-cadherin levels.
“Heat shocking WT larvae did not increase occludin, claudin 5, or E-cadherin levels (Supplemental Figure 4, A-C).”
Quote in the paperSubstantial claim - Interpretationown data
Snail1 is sufficient to disrupt brain endothelial tight junctions.
“Snail1 is sufficient to disrupt brain endothelial tight junctions.”
Quote in the paperSubstantial claim - Interpretationown data
Snail1 is necessary to disrupt brain endothelial tight junctions.
“Snail1 is necessary to disrupt brain endothelial tight junctions.”
Quote in the paperSubstantial claim - Interpretationown data
MAPK signaling contributes to SNAI1 expression.
“Contribution of MAPK signaling in SNAI1 expression.”
Quote in the paperSubstantial claim - Interpretationown data
GBS factors contribute to SNAI1 expression.
“GBS factors contribute to SNAI1 expression.”
Quote in the paperSubstantial claim - Interpretationown data
Snail1 contributes to GBS-BBB penetration in vivo.
“Contribution of Snail1 to GBS-BBB penetration in vivo.”
Quote in the paperSubstantial claim
- May combine two claims. 20
- The quote only partly states this claim. 5
- Classified as probably not a substantial claim. 13
May combine two claims.
Findingown dataSNAI1 induction occurs with 3 different GBS clinical serotypes but not with nonpathogenic bacterial strains or S. aureus.
“SNAI1 induction occurred following infection with 3 different GBS clinical serotypes (Figure 1D), but not with nonpathogenic bacterial strains or S. aureus, which is not classically associated with meningitis”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims. The quote only partly states this claim.
Findingown dataSnail1 overexpression decreases ZO-1, occludin, and VE-cadherin levels and disrupts tight junction integrity in hBMECs.
“Induction of Snail1 resulted in decreased levels of ZO-1, occludin (Figure 4, A, C, and D), and VE-cadherin (Supplemental Figure 2, C and D).”
- Also stated in: § Snail1 is necessary and sufficient to disrupt tight junctions. · plain
“Overexpression was also coupled with a loss in tight junction integrity as visualized by ZO-1 staining (Figure 4E).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Also stated in: § Snail1 is necessary and sufficient to disrupt tight junctions. · plain
May combine two claims. The quote only partly states this claim.
Findingown dataCell wall extract from Δiag mutant reduces SNAI1 induction compared with WT, and double capsule/Δiag mutant reduces further.
“We observed that cell wall extract from the Δiag mutant resulted in significantly reduced levels of SNAI1 transcripts when compared with those of cell wall extracts prepared from WT GBS cell wall extract.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
The quote only partly states this claim.
Findingown dataSNAI1 knockdown rescues occludin transcript and protein levels in GBS-infected hBMECs.
“Additionally, whereas GBS infection resulted in decreased occludin transcript levels in shSNAI1-hBMECs, GBS infection led to a modest rescue of occludin in doxycycline-treated shSNAI1-hBMECs (Figure 3D).”
- Also stated in: § Snail1 is necessary and sufficient to disrupt tight junctions. · plain
“Furthermore, doxycycline treatment of cells resulted in a rescue of occludin (Figure 3, E and F).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Also stated in: § Snail1 is necessary and sufficient to disrupt tight junctions. · plain
May combine two claims.
Findingown dataGBS infection increases SNAI1 transcript levels in hBMECs and bEND3 cells.
“Quantitative PCR (qPCR) analysis revealed that the SNAI1 transcript was significantly increased in infected cells compared with uninfected control cells (Figure 1, B and C).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims.
Findingown dataHeat-killed GBS does not induce SNAI1, but formalin-fixed GBS induces SNAI1 transcripts comparably to live bacteria.
“Interestingly, HK GBS did not induce SNAI1, however formalin-fixed GBS resulted in the induction of SNAI1 transcripts comparable to the levels seen in infection with live bacteria (Figure 5A).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims. The quote only partly states this claim.
Findingown dataGBS infection decreases transcript and protein levels of ZO-1, occludin, claudin 5, and VE-cadherin in hBMECs.
“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.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims. The quote only partly states this claim.
Findingown dataGBS infection causes up to 90% mortality, high bacterial loads in blood and brain, aberrant swimming, cerebral swelling and edema in adult zebrafish.
“When zebrafish were injected with WT GBS or a PBS vehicle control, we observed up to 90% mortality in GBS-infected fish (Figure 7A) and high bacterial loads in the blood and brain (Supplemental Figure 3, A and B).”
- Also stated in: § Contribution of Snail1 to GBS-BBB penetration in vivo. · plain
“Furthermore, GBS-infected fish exhibited cerebral swelling and edema typical of CNS infection (Figure 7B).”
Quote in the paperSubstantial claim- Also stated in: § Contribution of Snail1 to GBS-BBB penetration in vivo. · plain
May combine two claims.
Study design and conductown dataControl hBMEC-TRE cell line does not show changes in Snail1, ZO-1, occludin or permeability.
“No changes in Snail1, ZO-1, or occludin were observed in a control cell line, which contained all the elements of the overexpression line except for the Snail1 ORF (hBMEC-TRE) (Supplemental Figure 2E).”
- Also stated in: § GBS infection disrupts tight junctions in brain endothelium. · plain
“The control cell line (hBMEC-TRE) did not exhibit any permeability (Supplemental Figure 2F).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
- Also stated in: § GBS infection disrupts tight junctions in brain endothelium. · plain
May combine two claims.
Interpretationown dataA GBS surface factor initiates SNAI1 transcription and is susceptible to heat denaturation.
“This result suggests that a GBS surface factor initiates SNAI1 transcription and that this surface factor is susceptible to heat denaturation.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims.
Study design and conductown dataDoxycycline treatment of control hBMECs does not change tight junction proteins or Snail1, and does not inhibit GBS growth.
“No changes in tight junction proteins or Snail1 were observed in doxycycline-treated control hBMECs (Supplemental Figure 2A), nor did doxycycline inhibit GBS growth at the concentration used (Supplemental Figure 2B).”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims.
Interpretationown dataBacterial LTA, TLR2 activation, and MEK1/2/ERK1/2 signaling lead to SNAI1 induction.
“Taken together, these data suggest that bacterial LTA, TLR2 activation, and signaling through MEK1/2/ ERK1/2 lead to SNAI1 induction.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims.
Study design and conductown dataGBS infection does not increase cell death or alter monolayer density in hBMECs, so tight junction staining differences are not due to cell death.
“Further, we observed no difference in cell monolayer density or cell viability, as measured by Trypan blue staining during infection (Supplemental Figure 1E), suggesting that the observed differences in tight junction staining were not a result of increased cell death.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
Classified as probably not a substantial claim.
PremisecitationAdult zebrafish have been used to model Streptococcus iniae and GBS.
“Adult zebrafish have been used to model Streptococcus iniae and, recently, GBS (32,33).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [32] source
Not checked in the free run.
- cites [33] Phelps, 2009, Curr Protoc Microbiol.
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
PremisecitationZebrafish Snail1 homolog Snail1a is required for EMT and neural crest development.
“As the zebrafish Snail1 homolog Snail1a is also required for EMT and neural crest development (58), this model was more amenable to the use of siRNA silencing to globally knock down expression of Snail1a.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [58] Thisse, 1993, Development. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationActivation of ERK1/2 results in increased ZO-1 degradation during TBI, decreased ZO-1 and claudin 5 in HIV, and reduced occludin and claudin 5 in ischemic stroke.
“Activation of ERK1/2 results in increased ZO-1 degradation during traumatic brain injury (51), decreased ZO-1 and claudin 5 expression in brain endothelium during HIV infection (52,53), and reduced occludin and claudin 5 expression in BBB endothelium during ischemic stroke (54).”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationMAPK signaling pathways modulate expression of tight junction proteins and alter molecular composition within tight junction complexes.
“MAPK signaling pathways modulate the expression of tight junction proteins and alter the molecular composition within tight junction complexes (49).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [49] source
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
PremisecitationSnail1 is a global transcriptional repressor of tight junctions and plays an important role in EMT during development.
“Snail1 is a global transcriptional repressor of tight junctions (22) and plays an important role in the epithelial-tomesenchymal transition (EMT) during development (26).”
Quote in the paperSubstantial claim0 of 2 sources checkedClassified as probably not a substantial claim.
PremisecitationLTA is a known TLR2 ligand.
“a known TLR2 ligand (30).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [30] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenInfection experiments in the larval zebrafish model were performed in a blinded fashion.
“Infection experiments were performed in a blinded fashion.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataIn brain endothelium, Snail1 induction is initiated by TLR2 and involves the ERK pathway of MAPK signaling cascade.
“Similarly, our results suggest that in brain endothelium, Snail1 induction is initiated by TLR2 and involves the ERK pathway of the MAPK signaling cascade.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSnail1 can downregulate claudin 1 and ZO-1 without affecting their transcription.
“However, Snail1 can also downregulate claudin 1 and ZO-1, without affecting their transcription (22).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [22] Ohkubo, 2004, J Cell Sci. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataExperiments were performed at least 3 times in triplicate.
“Experiments were performed at least 3 times in triplicate.”
- Also stated in: § Adult zebrafish model of GBS infection. · plain
“Experiments were performed at least 3 times in triplicate.”
- Also stated in: § Adult zebrafish model of GBS infection. · plain
“Experiments were performed at least 3 times in triplicate.”
Quote in the paperNot a substantial claim- Also stated in: § Adult zebrafish model of GBS infection. · plain
Classified as probably not a substantial claim.
PremisecitationTLR2 contributes to pathogenesis of GBS meningitis.
“As TLR2 contributes to the pathogenesis of GBS meningitis (5) and downstream signaling of TLRs activate the MAPK pathway, we hypothesized that treatment of hBMECs with the TLR2 agonist Pam3CSK4 (P3C) would induce SNAI1 in hBMECs.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [5] source
Not checked in the free run.
May combine two claims.
Interpretationown dataInfection with multiple meningitis-associated pathogens induces SNAI1, which is necessary and sufficient to disrupt tight junctions in brain endothelium.
“Here, we demonstrate that infection with multiple pathogens associated with meningitis results in the induction of SNAI1, which we demonstrate to be necessary and sufficient to disrupt tight junctions in brain endothelium.”
Quote in the paperSubstantial claim- Rests on an assumption: hBMECs are a valid model of human brain microvascular endothelium.
May combine two claims.
PremisecitationGBS pilus protein PilA and Srr-1 interact with host ECM to promote BBB interactions and meningitis.
“Recently, we have demonstrated that the GBS pilus protein PilA and Srr-1 interact with components of the host extracellular matrix (ECM) to promote BBB interactions and the development of meningitis (8,9).”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOther meningeal pathogens bind ECM components and receptors to mediate bacterial-BBB interactions.
“Other meningeal pathogens, such as Streptococcus pneumoniae (SPN), E. coli K1, Neisseria meningitidis, and Haemophilus influenza type B (HiB, also bind ECM components and ECM receptors (e.g., integrins and laminin receptor) to mediate bacterial-BBB interactions (12)(13)(14)(15).”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataRepresentative data from 1 of 2 independent experiments are shown.
“Representative data from 1 of 2 independent experiments are shown.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataInhibition of Snail1 impacts expression of junctional components and reduces GBS virulence in zebrafish larvae.
“Inhibition of Snail1 impacts expression of junctional components and reduces GBS virulence in zebrafish larvae.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenData represent 3 independent experiments combined.
“Data represent 3 independent experiments combined.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
hBMECs are a valid model of human brain microvascular endothelium.
“Immortalized hBMECs (a gift of Kwang Sik Kim and Monique Stins, Johns Hopkins University, Baltimore, Maryland, USA) were cultured in RPMI 1640 containing 10% FBS, 10% NuSerum, and 1% nonessential amino acids as described previously (5,6,64).”
The paper relies on this without support.
The Tg(HSP:dn-snai1a) zebrafish line functions as a dominant-negative inhibitor of endogenous snai1a.
“We generated Tg(HSP:dn-snai1a) animals that function as a dominant-negative inhibitor of endogenous snai1a.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Group B streptococcus promotes expression of a transcriptional repressor (Snail1) in CNS vascular endothelial cells, causing down-regulation of tight junction proteins (occludin, ZO-1, and claudin-5) and a loss of barrier integrity.
“For example, group B streptococcus, a microbe that causes bacterial meningitis, promotes expression of a transcriptional repressor (Snail1) in CNS vascular endothelial cells, causing down-regulation of tight junction proteins (occluding, ZO-1, and claudin-5) and a loss of barrier integrity (149).”
From The anatomy and immunology of vasculature in the central nervous system (2019) · cites this paper as [149]
The citation lists haven’t been fetched yet
References 74 · Cited by 145
Research works printed but not linked: 4
- Betz, 1992, NIDA Res Monogr., 120, 54
- Schneeberger, 1992, Am J Physiol., 262, L647
- Phelps, 2009, Curr Protoc Microbiol., Chapter 9, Unit 9D.1
- Li, 2011, Asian Pac J Cancer Prev., 12, 2575
- Authors
- Kim, B. J., Hancock, B. M., Bermudez, A., Cid, N. D., Reyes, E., van Sorge, N. M., Lauth, X., Smurthwaite, C. A., Hilton, B. J., Stotland, A., Banerjee, A., Buchanan, J., Wolkowicz, R., Traver, D., & Doran, K. S.
- Affiliations
- San Diego State University (US)
- University of California San Diego (US)
- Corresponding author
- Doran, Kelly S.
- ORCID
- Venue
- Journal of Clinical Investigation
- Published
- 2015-05-11
- Volume
- 125
- Issue
- 6
- Pages
- 2473-2483
- ISSN
- 0021-9738 (ISSN-L)
- PMC
- PMC4497739
- OpenAlex
- W1495151309
- Semantic Scholar
- 6c19f40e46977dac6837c4d020a69565167aedf3
- Cited by
- 145
- References
- 74
- Citations by year
- 2026: 11 · 2025: 13 · 2024: 16 · 2023: 8 · 2022: 18 · 2021: 17 · 2020: 14 · 2019: 14 · 2018: 5 · 2017: 13 · 2016: 10 · 2015: 6
- FWCI
- 6.06
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Journal of Clinical Investigation (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Funders
- American Heart Association (14PRE18690037)
- Achievement Rewards for College Scientists Foundation
- National Institutes of Health
- University of California, San Diego
- National Institute of General Medical Sciences (R25GM058906)
- National Institute of Neurological Disorders and Stroke (R01NS051247)
- Topics
- Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
- Life Sciences › Immunology and Microbiology › Microbiology › Bacterial Infections and Vaccines
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Neonatal and Maternal Infections
- Keywords
- SNAI1, blood-brain barrier disruption, bacterial meningitis, Streptococcus agalactiae, tight junctions, endothelial cells, ERK signaling, bacterial cell wall, ZO-1, claudin-5, occludin, bacterial translocation, zebrafish, neonatal meningitis
- MeSH terms
- Snail Family Transcription Factors, Animals, Blood-Brain Barrier, Cells, Cultured, Gene Expression Regulation, Humans, Streptococcal Infections, Streptococcus agalactiae, Transcription Factors, Zebrafish, Tight Junctions, Mitogen-Activated Protein Kinase 1, MAP Kinase Signaling System, Zebrafish Proteins, Mitogen-Activated Protein Kinase 3, Claudin-5, Zonula Occludens-1 Protein
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.
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