Low Ply expression promotes pneumococcal trafficking across BBB.

<p>Balb/c mice were infected via i.v. route with 10⁶ CFU of WT, WT:Ply-High or WT:Ply-Low SPN strain and at 14 h p.i. animals were sacrificed. PBS injected mice served as control. <b>A.</b> Confocal micrographs depicting heterogeneous Ply expression (red) within WT SPN population (blue) in the blood of infected mice. Blood smears were stained with Anti-Ply antibody and DAPI. Scale bar, 5 μm. <b>B.</b> Ratio of transcript levels of Ply in brain to blood of mice infected with WT SPN. Transcript levels were normalized to 16S rRNA and expressed as fold change compared to Ply transcript levels of WT SPN grown <i>in vitro</i> in THY medium. <b>C.</b> Kaplan-Meier survival curve of mice (n = 5 per group) following i.v. injection with different SPN strains. Experiments were repeated twice and one representative graph is shown. Statistical analysis was performed using Log-rank test. <b>D.</b> Net growth of SPN strains in the blood of infected mice (n = 5 per group) at indicated time points post infection. Experiments were repeated twice and one representative graph is shown. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, non-significant. <b>E.</b> Quantification of bacterial counts (CFU) in various tissue homogenates and blood of mice infected with different Ply expressing SPN strains. Each dot represents one mouse; black bars show average values. n = 12 per group (2 experiments with n = 6 per group). Statistical analysis for each tissue was individually performed using non-parametric test (Mann-Whitney test). <i>p</i> values are mentioned in the graph. <b>F—H.</b> Ratio of bacterial CFU in brain to blood <b>(F)</b>, lungs to blood <b>(G)</b> and spleen to blood <b>(H)</b> of individual infected mice. Each dot represents one mouse; black bars show average values. n = 12 per group (2 experiments with n = 6 per group). Statistical analysis was performed using non-parametric test (Mann-Whitney test). <i>p</i> values are mentioned in the graph. <b>I.</b> Transcript abundance of pro-inflammatory cytokines in total RNA isolated from brains of mice that were injected with PBS or infected with different SPN strains. Transcript levels were normalized to 18S rRNA and expressed as fold change compared to control mice. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, nonsignificant. <b>J.</b> Histopathology of H & E stained representative brain tissue samples of control (PBS injected) or mice infected with different strains of SPN. Scale bar, 100 μm.</p

Heterogeneous Ply expression is a serotype independent phenomenon.

<p><b>A.</b> Expression of Ply on pneumococcal cell surface was assessed by flow cytometry following staining with Anti-Ply Ab. Different SPN strains used for the study included; D39 (serotype 2), TIGR4 (serotype 4), Tupelo (serotype 14) and A60 (serotype 19F). Pink curve depicts unstained SPN cells while blue curve represents antibody stained SPN cells. Experiments were repeated thrice and representative histograms are shown. <b>B.</b> Percentage of Ply positive cells in different SPN serotype strains as analyzed by flow cytometry. Data is represented as mean ± SD of triplicate experiments. Statistical analysis was performed using one-way ANOVA (Tukey’s Multiple comparison test). ns, non-significant; **<i>p<</i>0.005.</p

Damage to endosomal membrane by Ply triggers autophagy mediated clearance of SPN in brain endothelium.

<p><b>A.</b> Intracellular survival efficiency of WT SPN, Δ<i>ply</i> mutant and Δ<i>ply</i>:pPly strains of R6 (serotype 2) in hBMECs were calculated as percent survival at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using one-way ANOVA (Tukey’s multiple comparison test). *<i>p</i><0.05; ***<i>p</i><0.001. <b>B.</b> Confocal micrographs showing association of Gal8 with GFP expressing WT and Δ<i>ply</i> SPN strains at 3 h p.i. in hBMECs. Arrowhead designates bacteria shown in insets. Event localized at z-stack no. 6 and 5 out of 10 and 11 for WT and Δ<i>ply</i>, respectively. Scale bar, 5 μm. <b>C.</b> Percent co-localization of Gal8 with WT and Δ<i>ply</i> SPN strains at indicated time points post-infection. n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate hBMEC cultures. <b>D.</b> Confocal micrograph showing association of WT SPN (blue) with Gal8 (red), NDP52 (pink) and LC3 (green) at 3 h p.i.. hBMECs stably expressing GFP-LC3 were infected with SPN and stained with anti-Gal8 Ab, anti-NDP52 Ab and DAPI. Arrowhead designates bacteria shown in insets. Event localized at z-stack no. 4 out of 9. Scale bar, 5 μm. <b>E.</b> Fluorescent line scan across the yellow line in the merged inset in “<b>D</b>”, depicting co-localization of SPN with Gal8, NDP52 and LC3. <b>F.</b> Quantification of co-localization of LC3 with Gal8 positive SPN in hBMECs at 3 h p.i. Data are presented as mean ± SD of triplicate experiments. n ≥ 50 bacteria per coverslip. <b>G.</b> Western blot demonstrating knock-down in expression of Gal8 in hBMEC cultures following transfection with a targeted siRNA against <i>LGALS8</i> (siGal8). GAPDH served as loading control. Relative ratio of Gal8 to GAPDH band intensities (normalized to siControl) is mentioned on top of the blot. <b>H.</b> Quantification of LC3 co-localization with WT SPN under Gal8 knockdown conditions using siGal8. Cells transfected with siScrambled served as control (siControl). n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using Students t-test. ***<i>p<</i>0.001. <b>I.</b> Intracellular survival of WT SPN in hBMECs following transfection with siGal8 or siControl at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate hBMEC cultures. Statistical analysis was performed using two-way ANOVA (Bonferroni test). ns, non-significant, *<i>p</i><0.05, **<i>p</i><0.005. <b>J–L.</b> Transmission electron micrograph depicting WT SPN <b>(J)</b> in vacuolar compartments inside hBMECs. Scale bar, 1 μm. Zoomed in view of the boxed area in “<b>J</b>” and “<b>K</b>” are shown in “<b>K</b>” (scale bar, 0.2 μm) and “<b>L</b>”, respectively. Arrow in “J” points towards SPN sequestered in a vacuole while arrows in “L” show disruptions in the PCV membrane.</p

Differential Ply expression governs the spatio-temporal location, survival and trafficking of SPN across BBB.

<p><b>A.</b> Flow cytometry of a recombinant SPN strain with <i>ply</i> gene transcriptionally fused with <i>gfp</i> to monitor variation in Ply expression among isogenic SPN population. <b>B.</b> Confocal micrographs depicting heterogeneous Ply expression within a population of <i>in vitro</i> grown SPN culture. GFP expressing SPN (<i>hlpA</i>-GFP integrated in genome) was probed with Anti-Ply Ab. Scale bar, 1 μm. <b>C—D.</b> Quantification of different population subsets of intracellular WT:Ply-High and WT:Ply-Low SPN strains at 3 h <b>(C)</b> and 9 h <b>(D)</b> p.i. for association with Gal8, Ubq and LC3. n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test). *<i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.001. <b>E.</b> Intracellular location of WT:Ply-High and WT:Ply-Low SPN strains at 3 and 9 h p.i. in hBMECs. Gal8⁺ Ubq^<b>−</b>LC3^-, Gal8⁺ Ubq^<b>−</b>LC3⁺, Gal8⁺ Ubq⁺ LC3⁺ and Gal8^<b>–</b> Ubq^<b>−</b>LC3⁺ constituted vacuolar SPN population, while Gal8^<b>–</b> Ubq⁺ LC3⁺ and Gal8^<b>–</b> Ubq⁺ LC3^<b>–</b> constituted vacuolar escaped SPN population. n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate hBMEC cultures. Statistical analysis was performed using two-way ANOVA (Bonferroni test). *<i>p</i><0.05, **<i>p</i>< 0.005. <b>F.</b> Comparison of intracellular survival efficiencies between WT:Ply-High and WT:Ply-Low strains in hBMECs expressed as percent survival at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test); *<i>p</i> <0.05, ***<i>p</i> <0.001. <b>G.</b> Comparison of egression efficiencies of WT:Ply-High and WT:Ply-Low SPN strains across confluent hBMEC monolayers at indicated time points. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, non-significant, **<i>p</i>< 0.005.</p

Degradation of cytosolic SPN via autophagy–dependent or independent pathway is reliant on Ply induced ubiquitination.

<p><b>A, C.</b> Representative time-lapse montage for degradation of cytosolic SPN following escape from autophagosomes in LC3-dependent <b>(A)</b> or independent manner <b>(C)</b>. hBMECs stably expressing LC3-GFP (green) and Ubq-mStrawberry (red) were infected with DRAQ5 stained WT SPN (white). Live imaging of infected cells was performed for extended hours beginning at 6 h p.i. under a confocal microscope and time-lapse series with intervals of 60 min <b>(A)</b> or 50 min <b>(C)</b> are represented. The stills in “<b>A</b>” and “<b>C</b>” correspond to movies shown as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007168#ppat.1007168.s010" target="_blank">S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007168#ppat.1007168.s011" target="_blank">S4</a> Movies, respectively. Scale bar, 2 μm. <b>B, D.</b> Temporal quantification of ubiquitin, LC3 and SPN fluorescence intensities either relative to fluorescence in the cytosol (Cyt) (for LC3 and Ubq) or fluorescence signal of hBMEC nuclei (Nuc) (for SPN). Relative mean intensity for LC3 (I_PCV/I_Cyt) in case of ubiquitinated SPN displayed values > 1.0, depicting LC3-dependent killing of SPN <b>(B)</b>. Ubiquitinated SPN that is degraded in LC3 independent manner, displayed relative mean intensity value ~ 1.0 for LC3 <b>(D)</b>. In both cases relative mean intensity values for bacteria (I_SPN/I_Nuc) remains > 1.0 for substantially long period of time before finally disappearing. <b>E.</b> Percentage of ubiquitinated SPN undergone LC3-dependent or independent killing as examined by time-lapse imaging of hBMECs stably expressing GFP-LC3 and Ubq-mStrawberry following infection with DRAQ5 stained SPN. n = 41. <b>F.</b> Intracellular survival percentages of WT SPN following treatment with MG132 (10 μM) at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test). ns, non-significant, ***<i>p</i><0.001.</p

Phenotypic heterogeneity of SPN inside brain endothelium.

<p>Confocal images of different population of WT SPN inside hBMECs at 6 h p.i. <b>(A)</b> Gal8⁺ Ubq^<b>–</b>LC3^<b>–</b>, <b>(C)</b> Gal8⁺ Ubq^<b>−</b>LC3⁺, <b>(E)</b> Gal8⁺ Ubq⁺ LC3⁺, <b>(G)</b> Gal8^<b>–</b> Ubq⁺ LC3⁺, <b>(I)</b> Gal8^<b>–</b> Ubq⁺ LC3^<b>–</b>, <b>(K)</b> Gal8^<b>–</b> Ubq^<b>−</b>LC3⁺. hBMECs stably expressing Gal8-YFP were infected with WT SPN (blue) and stained with anti-LC3 Ab (pink) and anti-Ubq Ab (red). Fluorescent line scans across the yellow lines in the merged insets in “<b>A, C, E, G, I</b> and <b>K</b>”, depicting association of SPN with Gal8, Ubq and LC3 are given in “<b>B, D, F, H, J</b> and <b>L</b>”, respectively.</p

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

<div><p>Infection of the genitourinary tract with Group B <i>Streptococcus</i> (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs <i>ex vivo</i> resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.</p></div

Intra-amniotic injections of GBS membrane vesicles cause tissue damage in the chorio-decidua.

<p>On E14.5 each fetus was injected with MVs or BSA encapsulated liposomes or PBS in the amniotic sac and after 24 h chorio-decidua were collected. Paraffin sections were stained with haematoxylin and eosin (H&E) and for collagen using Picrosirius red (scale bar; 200 μm) or processed for detection of apoptosis by TUNEL (scale bar; 10 μm). Arrows indicate disruptions in the epithelial cell lining (in H&E images) and collagen fiber disintegration (in collagen staining images) in MV injected tissues. Chorion, amnion and amniotic fluid (AF) are designated in the H&E image of PBS injected tissue. ‘*’ designates basement membrane in collagen staining images.</p

Characterization and biological activity of membrane vesicles isolated from GBS strain A909.

<p><b>A.</b> Relative abundance of different fatty acids in MVs and whole bacterial cells analyzed using a GC-MS. Compounds were identified by comparison of the mass spectra with a library. Total fatty acids and other small molecules found in intact GBS cells were considered as 100% and the relative amounts of the same for MVs were calculated accordingly. Experiments were performed thrice. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, non-significant. <b>B.</b> SDS-PAGE of proteins packaged in MVs. 150 μg of MV protein was precipitated with TCA (10%) and separated on 12% SDS-PAGE. Selected protein bands (indicated by arrow) were excised from the gel, trypsinized and the resulting peptides were analyzed by MALDI mass spectrometry followed by identification with Mascot search engine. The IDs of the corresponding proteins are given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005816#ppat.1005816.t001" target="_blank">Table 1</a>. <b>C.</b> PCR amplification of <i>cylE</i> and <i>cfb</i> genes in DNA isolated from GBS cells or MVs. Lane 1: DNA ladder (1 Kb). The corresponding size of bands in Kb are marked. <b>D.</b> Cytotoxicity of MVs to HeLa cells. HeLa cells were incubated with different conc. of MVs or BSA entrapped liposomes (10 to 300 μg of protein/ml) for 24 h and cell viability was determined by MTT assay. 1% Triton X-100 solution was used as a positive control. Experiments were performed three times (each in triplicate) and results are expressed as fold change in cell viability with respect to highest value. Bars are mean ± standard deviation. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ***<i>p</i> < 0.001; ns, non-significant. <b>E.</b> Internalization of GBS MVs in HeLa cells. FITC labeled MVs were incubated with HeLa cells for 6 h. Extracellular MVs were stained with anti-FITC Ab, followed by AlexaFluor 555 conjugated secondary antibody. Internalized MVs showed green fluorescence (white arrows) while the extracellular MVs demonstrated red fluorescence. Scale bar; 5 μm.</p

Intra-amniotic injections of GBS membrane vesicles cause inflammatory changes in the chorio-decidua.

<p><b>A.</b> On E14.5, each fetus was injected with either PBS or membrane vesicles (MVs) in the amniotic sac. 24 h after injections, chorio-decidua were collected. Paraffin sections were stained using an antibody against the macrophage marker F4/80 Ab. Dark brown staining is for F4/80, indicating presence of macrophages.–ve Control implicates no non-specific binding of the secondary Ab. Scale bar; 200 μm. <b>B.</b> mRNA levels of IL-1β, TNF-α, IFN-γ, KC and IL-6 was determined by qRT–PCR on total RNA isolated from decidua, 24 h post injection with MVs (n = 8). Transcript levels were normalized to 18s rRNA and expressed as fold change compared with tissues injected with PBS only. Statistical analysis was performed using two-way ANOVA (Bonferroni test); *<i>P</i> < 0.05.</p