Figure 5 – Inhibition of GSL synthesis and CtxB influences Hc- Mφ association.

(A) Inhibition of GSL expression decreases fungal association to J774.16 Mφ (p<0.05). Mφs treated with the CerGlcT inhibitor P4r (active isomer) for 3 days have a 70% inhibition of ganglioside expression whereas exposure to P4s (inactive isomer) had no effect (data not shown). Treated Mφs were incubated with GFP-Hc and the association index determined by FACS analysis. (B) Kinetics of Hc- Mφ adhesion measured by OT. Treatment of Mφs with P4r (Δ) results in decreased kinetics of adhesion compared with control systems (■). Characteristic time (τ) required for adhesion is shown. (C) TLC showing major gangliosides produced by J774 cells. GM1 and GD1a are indicated and were detected by immunoassays using specific mAbs (data not shown). (D) GFP-Hc yeast cells with or without opsonization with MAb 7B6 were incubated for 45 minutes with WT or B4galnt1^−/− peritoneal Mφs. Samples were analyzed by flow cytometry. (E) Hc yeasts were incubated with J774.16 Mφs previously treated with CtxB (1μg/ml). Binding of CtxB to Mφ cell surface inhibited fungal association (CtxB/ Hc). Pre-treatment of yeasts with mAbs to HSP60 reversed the effect of CtxB (CtxB/Ab- Hc). Association percentage values normalized by the control are shown (above bars). (F) Hc yeast were co-incubated with oligo-GM1 and J774.16 Mφs. Inhibition of fungal association by oligo-GM1 was dose dependent. The experiments were performed at least twice with similar results.