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. 2018 May 1;128(5):1985-1999.
doi: 10.1172/JCI97043. Epub 2018 Apr 9.

Group B Streptococcus Exploits Vaginal Epithelial Exfoliation for Ascending Infection

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Free PMC article

Group B Streptococcus Exploits Vaginal Epithelial Exfoliation for Ascending Infection

Jay Vornhagen et al. J Clin Invest. .
Free PMC article

Abstract

Thirteen percent of pregnancies result in preterm birth or stillbirth, accounting for fifteen million preterm births and three and a half million deaths annually. A significant cause of these adverse pregnancy outcomes is in utero infection by vaginal microorganisms. To establish an in utero infection, vaginal microbes enter the uterus by ascending infection; however, the mechanisms by which this occurs are unknown. Using both in vitro and murine models of vaginal colonization and ascending infection, we demonstrate how a vaginal microbe, group B streptococcus (GBS), which is frequently associated with adverse pregnancy outcomes, uses vaginal exfoliation for ascending infection. GBS induces vaginal epithelial exfoliation by activation of integrin and β-catenin signaling. However, exfoliation did not diminish GBS vaginal colonization as reported for other vaginal microbes. Rather, vaginal exfoliation increased bacterial dissemination and ascending GBS infection, and abrogation of exfoliation reduced ascending infection and improved pregnancy outcomes. Thus, for some vaginal bacteria, exfoliation promotes ascending infection rather than preventing colonization. Our study provides insight into mechanisms of ascending infection by vaginal microbes.

Keywords: Bacterial infections; Infectious disease; Reproductive Biology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. GBS stimulates exfoliation and disrupts vaginal epithelial cell barrier function in vitro.
(A) hVECs were infected with WT GBS for 0, 16, or 24 hours and stained with 10% crystal violet for 30 minutes. Loosely adherent cells were removed by centrifugation, and crystal violet staining intensity was measured. Data were normalized to mock-infected controls (n = 3; **P < 0.005 and P = 0.08, by ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (B) hVECs were infected with WT GBS for 24 hours and analyzed by scanning electron microscopy. Images show hVEC detachment 24 hours after infection with control PBS (mock) or WT GBS. Scale bars: 10 μm. (C) The barrier function of hVEC monolayers was monitored in real time using ECIS. Infection with WT GBS led to a disruption in barrier function as determined by the decrease in resistance of the infected monolayers compared with the uninfected control (n = 3; data represent the mean). (D) hVECs grown on Transwells were infected with WT GBS for 24 hours or treated with 0.1% Triton X-100, which was used as a positive control. After 24 hours, fluorescein dye was added to the apical compartment of the Transwell, and migration of the dye to the basal compartment was measured after 1 hour (n = 3; ****P < 0.00005, by ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (E) hVECs grown on Transwells were either mock infected or infected with WT GBS for 24 hours. At 0, 16, and 24 hours after infection, the basal compartment of each well was sampled and stained with SYTO9 nucleic acid to enumerate bacteria (n = 3; **P < 0.005, by ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). RFU, relative fluorescence units.
Figure 2
Figure 2. GBS stimulates exfoliation and disrupts vaginal epithelial cell barrier function in vivo.
(A) Female WT C57BL6/J mice were vaginally inoculated with approximately 108 CFU of either WT GBS or an equal volume of control PBS (n = 3/group). Vaginal tissues were analyzed by scanning electron microscopy, and bacterial burden was assessed in vaginal and uterine tissues. Images show significant vaginal epithelial exfoliation 72 and 96 hours after inoculation with WT GBS and not with control saline. Scale bars: 100 μm. (B) Exfoliated vaginal epithelial cells 24, 48, 72, and 96 hours after inoculation were quantified in a blinded fashion (n = 3 images/2 tissues/group; **P < 0.005 and ****P < 0.00005, by ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (C) High-magnification images of the vaginal epithelium show that GBS was associated with exfoliated epithelial cells. Scale bars: 10 μm; Original magnification, ×1900 (enlarged inset). Images are from 1 of at least 3 experiments. (D) Nanoparticle penetration into mouse vaginal epithelia in control saline– or WT GBS–treated animals 24 and 96 hours after vaginal inoculation, respectively. Nuclei were stained with DAPI and are shown in blue, PEGylated nanoparticles (120 nm diameter) are shown in white, and red arrows indicate intraepithelial nanoparticles. Images are from 1 of at least 2 experiments. Scale bars: 100 μm. Quantitative measurements were calculated using the equation: ([mean area of intraepithelial nanoparticle coverage]/[mean epithelial area]) × 10,000 ± SD. (E) GBS penetration into mouse vaginal epithelia in control saline– or WT GBS–treated animals 24 and 96 hours after vaginal inoculation. Nuclei were stained with DAPI and are shown in blue, GBS are shown in white, and yellow arrows indicate intraepithelial GBS. Images are from 1 of at least 4 experiments. Scale bars: 10 μm.
Figure 3
Figure 3. GBS induces EMT.
Flow cytometric analysis of surface E-cadherin (A) or N-cadherin (B) expression on GBS-infected hVECs compared with the mock (PBS) control (n = 3; **P < 0.005 and ***P < 0.0005, by 2-sided, unpaired t test; data represent the mean ± SEM). (C) Quantitative reverse transcription PCR analysis of EMT markers in GBS-infected hVECs compared with mock-treated controls (n = 3; **P < 0.005 and ****P < 0.00005, by 1-way ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (D) E-cadherin immunostaining in murine vaginal tracts 24 and 96 hours after vaginal inoculation with PBS or WT GBS, respectively. Images are from 1 of 3 experiments. Scale bars: 100 μm. (E) Flow cytometric analysis of surface E-cadherin on CD326+ mVECs 96 hours after vaginal inoculation with WT GBS or control PBS (n = 6 mice/group; *P < 0.05, by 2-sided, unpaired t test; data represent the mean).
Figure 4
Figure 4. GBS induces β-catenin signaling.
(A) Expression of β-catenin target genes in GBS-infected hVECs compared with expression in mock-treated controls 24 hours after infection (n = 3; **P < 0.005 and ****P < 0.00005, by 1-way ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (B) Localization of β-catenin (white) in GBS-infected hVECs compared with mock-treated controls 24 hours after infection (nuclei are stained with DAPI [blue]; overlap is shown in yellow). Images are from 1 of 3 experiments. Original magnification, ×100. (C) Expression of β-catenin target genes in murine vaginal tissues 96 hours after vaginal inoculation with WT GBS compared with expression in control PBS–treated tissues (n = 4/group; *P < 0.05, ***P < 0.0005, ****P < 0.00005, and P = 0.1, by 1-way ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (D) c-Myc immunostaining in murine vaginal tracts 96 hours after vaginal inoculation with PBS or WT GBS. Images are from 1 of 3 experiments. Scale bars: 100 μm. (E) Quantification of c-Myc immunostaining in murine vaginal tracts 96 hours after vaginal inoculation with WT GBS or control PBS (6 mice/group; *P < 0.05, by 2-sided, unpaired t test; data represent the mean). (F) Western blot (WB) for p-GSK3β in GBS-infected hVECs compared with mock-treated controls, 0 and 4 hours after infection. GAPDH was used as a loading control. Blots are from 1 of 4 experiments. (G) Quantification of p-GSK3β band intensity. The band intensity was first normalized to GAPDH and then to t0 of the corresponding treatment (n = 4/group; *P < 0.05, **P < 0.005, and P = 0.98, by 1-way ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (H) hVECs were left untreated or were treated for 16 or 24 hours with the β-catenin signaling inhibitor FH535 (15 μM) prior to WT GBS infection, and cell detachment was measured. Data were normalized to the uninfected controls (n = 3; *P < 0.05 and ***P < 0.0005, by ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (I and J) Flow cytometric analysis of surface E-cadherin (I) and N-cadherin (J) on mock-treated or WT GBS–infected hVECs, with or without FH535 pretreatment (n = 3; *P < 0.05, **P < 0.005, and P = 0.06; data represent the mean ± SEM by ANOVA followed by Sidak’s multiple comparisons test.
Figure 5
Figure 5. GBS activates integrin signaling.
(A) Flow cytometric plot of active β1 integrin (9EG7 antibody) on the surface of GBS-infected hVECs compared with mock-infected controls after 24 hours. Data are from 1 of 3 experiments. (B) Colorimetric assay of active integrin on the surface of mock-infected or WT GBS–infected hVECs (n = 3/group; ***P < 0.0005, by 2-sided, unpaired t test; data represent the mean ± SEM). (C) Western blots for p-FAK and p-AKT in GBS-infected hVECs compared with mock-treated controls, 0 and 4 hours after infection. GAPDH was used as a loading control. Blots are from 1 of 4 experiments. (D and E) Band intensity quantification for p-FAK (D) and p-AKT (E). Band intensity was first normalized to GAPDH as a loading control and then to t0 of the corresponding treatment (n = 4/group; *P < 0.05 and **P < 0.005, by 1-way ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM). (F) Active β1 integrin immunostaining in murine vaginal tracts 96 hours after vaginal inoculation with PBS, WT GBS, WT GBS with recombinant murine α1β1 integrin, or WT GBS with vehicle control. Images are from 1 of 3 experiments. Scale bars: 100 μm. (G) E-cadherin immunostaining in murine vaginal tracts 96 hours after vaginal inoculation of GBS with recombinant murine α1β1 integrin or vehicle control. Images are from 1 of 3 experiments. Scale bars: 100 μm. (H) Vaginal epithelial exfoliation in mice 96 hours after vaginal inoculation with WT GBS and treatment with vehicle control or recombinant murine α1β1 integrin. Scale bars: 100 μm. (I) Blinded quantification of exfoliated vaginal epithelial cells 96 hours after inoculation (n = 3 tissues/group; *P < 0.05, by 2-sided, unpaired t test; data represent the mean ± SEM).
Figure 6
Figure 6. Integrin-mediated epithelial exfoliation promotes ascending GBS infection.
(A) Bacterial burden in the vagina (n = 8; data represent the median). (B) Bacterial burden in the uterus (n = 8; *P < 0.05, by ANOVA followed by Sidak’s multiple comparisons test; data represent the median). (C) Bacterial burden in vaginal tissue from mice that were intravaginally treated with recombinant murine α1β1 integrin or control vehicle 96 hours after vaginal inoculation with WT GBS (n = 7–8; P = 0.629, by Mann-Whitney U test; data represent the median). (D) Bacterial burden in uterine tissue from mice that were intravaginally treated with recombinant murine α1β1 integrin or control vehicle 96 hours after vaginal inoculation with WT GBS (n = 8; **P < 0.05, by Mann-Whitney U test; data represent the median). (E) Pregnant female mice were vaginally inoculated with approximately 108 CFU of WT GBS and intravaginally treated with recombinant murine α1β1 integrin or vehicle control. Seventy-two hours after inoculation or at the first sign of preterm birth (vaginal bleeding and/or pups in cage), mice were euthanized, and the bacterial burden in vaginal tissue, uterine tissue, placental tissue, or fetal tissue was enumerated (n = 10–11; **P < 0.005, ***P < 0.0005, P = 0.605, and P = 0.052, by Mann-Whitney U test; data represent the median). (F) Pups with adverse birth outcomes (either in utero fetal demise or premature birth) from pregnant female mice that were vaginally inoculated with WT GBS and either intravaginally treated with recombinant murine α1β1 integrin (5 of 92 pups) or not (15 of 92 pups). Data represent the percentage of pups that had an adverse birth outcome (*P < 0.05, by 2-sided Fisher’s exact test. Bars represent % of pups with an adverse outcome out of total pups (indicated by n).
Figure 7
Figure 7. Model of GBS-induced epithelial exfoliation and ascending infection.
Upon GBS colonization, integrins are either directly or indirectly activated by GBS. Integrin activation induces phosphorylation of FAK, which in turn phosphorylates AKT, which phosphorylates GSK3β. As adherens junctions break down, β-catenin is released into the cytoplasm. In its dephosphorylated form, GSK3β marks β-catenin for degradation, thus preventing β-catenin stabilization, nuclear translocation, and signaling; however, when it is phosphorylated, it cannot mark β-catenin for degradation, leading to β-catenin stabilization and nuclear translocation. Once in the nucleus, β-catenin stimulates the expression of a variety of genes, including those that drive EMT and epithelial exfoliation. Rather than eliminating colonized GBS, epithelial exfoliation permits bacterial dissemination through the loss of barrier function. This leads to increased ascending infection, which increases the rates of adverse pregnancy outcomes and preterm birth.

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