CiaR regulation influences GBS intracellular trafficking in brain endothelial cells.

<p>A. hBMEC monolayers were infected with WT COH1 GFP expressing GBS for 2 hours (MOI = 10) and then stained with antibodies to Rab5, Rab7, and LAMP1 following the indicated time points as described in Materials and Methods. Representative images of triplicate experiments demonstrate co-localization (yellow) of GBS (green) with each marker (red), Scale Bar, 5μm. B, C, D. Percentages of co-localization of Rab5, Rab7, and LAMP1 with GFP expressing GBS WT and mutant strains after various time points post infection during antibiotic treatment. At least 100 cells containing intracellular GBS were counted for each time point in triplicate. Statistical analysis performed was a Two-way ANOVA with a Bonferroni’s multiple comparisons test and the data represents mean ± S.D. <i>p <</i> 0.05 *, <i>p <</i> 0.0005 ***, <i>p <</i> 0.00005 ****.</p

Adherence, invasion, and intracellular survival of GBS in brain endothelial cells are influenced by SAN_2180 and SAN_0039.

<p>A. Adherence to hBMEC by WT and mutant strains (MOI = 1). B. Invasion of hBMEC by WT and mutant strains (MOI = 1). C. Relative percentage of the hBMEC-associated GBS, WT and mutant strains, that had invaded the intracellular compartment (MOI = 1). D. Intracellular survival of WT and mutant GBS strains in hBMEC over time. Statistical analysis performed was a Two-way ANOVA with a Bonferroni’s multiple comparisons test and the data represents mean ± S.D. <i>p <</i> 0.05 *, <i>p <</i> 0.0005 ***, <i>p <</i> 0.00005 ****.</p

GBS SAN_2180 and SAN_0039 contribute to overall bacterial virulence.

<p>The recovered bacterial CFU in blood and brains were analyzed 72 h after intravenous injection with equal amounts of WT and mutant GBS strains into CD1 mice. Bacteria were enumerated on THA plates with serial dilutions and the bacterial colonies from the same dilution were distinguished by colonies PCR between WT and mutant strains. A. The percentage of recovered CFU in blood and brains from WT and the Δ2180 mutant strain. B. The percentage of recovered CFU in blood and brains from WT and the Δ0039 mutant strain. Statistical analysis performed was an unpaired t-test and the data represents mean ± S.D. <i>p <</i> 0.00005 ****.</p

GBS recovery from Lysosomes.

<p>A. Isolated lysosomes infected with WT COH1 GFP expressing GBS were subjected to staining with Lysotracker Red (0.5μM) and visualized using fluorescence microscopy. Scale Bar, 5 μm B. hBMEC were infected with WT or mutant GBS strains for 2 hours (MOI = 10) and subjected to lysosomal isolation by differential centrifugation after 1 or 12 hours post antibiotic treatment. Lysosomal pellets were plated to enumerate the amount of recovered viable CFU. Statistical analysis performed was a One-way ANOVA with a Tukey’s multiple comparisons test and the data represents mean ± S.D. <i>p <</i> 0.05 *, <i>p <</i> 0.005 **.</p

Morphology of GBS WT and mutant strains.

<p>A. Diagrammatic representation of the genetic locus surrounding SAN_2180 and SAN_0039 in the WT COH1 strain. Analysis of WT, Δ<i>ciaR</i>, Δ<i>2180</i> and Δ<i>0039</i> mutant strains by Gram staining (scale bar = 10 μm), B, and Scanning electron microscopy (top panel scale bar = 50 μm; bottom panel scale bar = 2μm), C.</p