Fig. 2: The cadD locus is required for GBS survival within human placental macrophages.

A–D Transmission electron microscopy analyses of human placental macrophages in co-culture with WT GB112 (A), ΔcadD isogenic mutant (B), or the ΔcadD:C complemented isogenic mutant (C), quantification of GBS cells in macrophages (macrophages from 3 independent biological replicates were analyzed) by TEM analyses (D) indicates cadD is required for intracellular persistence and replication (****P < 0.0001, one-way ANOVA with Tukey’s post hoc multiple corrections test). Magnification bar indicates 2 μm. E Analysis of phagocytosis of GBS by primary human placental macrophages. GBS cells were labeled with the fluorophore FITC and co-cultured with placental macrophages for 3–4 h to allow phagocytosis to occur. Extracellular bacterial fluorescence was quenched with trypan blue and fluorescence was measured as a proxy for intracellular bacteria (mean fluorescence indicates fluorescence-background fluorescence of untreated placental macrophages). Gentamicin protection assay evaluation of intracellular bacterial viability (F). D–F Lines indicate mean +/− standard error mean. Co-cultures of GBS and placental macrophages were performed with WT GB112 (pink circles or pink bars), ∆cadD isogenic mutant (purple squares or purple bars), and ∆cadD:C complemented mutant (blue triangles or blue bars); uninfected controls are indicated as white bars (*P = 0.0183, one-way ANOVA with Tukey’s post hoc multiple corrections test). G Analysis of human placental macrophage secretion of cytokines (IL-1β, IL-6, G-CSF, GM-CSF, MIP-1α, and MIP-1β) in response to GBS infection (*P < 0.0001, one-way ANOVA with Dunnet’s post hoc multiple corrections test. ^#P < 0.05 one-tailed, Student’s t test). All experiments were derived from three independent biological replicates (individual data points indicate macrophages derived different patient samples on different days). Bars indicate mean and error bars indicate +/− standard error mean.