Figure 3. Co-cultivation of Cn H99 and Hc GFP enhances pellicle formation by glycan transfer.
(a) Pellicle formation in HAM’s F-12 media was determined by measuring fungal metabolic activity using XTT colorimetric analysis. The initial inoculum for each well was 106 yeast cells, either all of one species or a 1:1 mix of 5 × 105 of Hc co-cultured with either Cn H99 or Cn cap59. Heat-killed Hc cells were used as background control and discounted from the readings. Co-cultures of Hc GFP + Cn H99 formed pellicles that were similar to biofilms produced by monospecies Cn H99. In contrast, Hc GFP + Cn cap59 and monospecies Hc GFP were extremely poor pellicle producers. (b) The reactivities of PS matrix of fungal pellicles were examined by ELISA using 2D10 IgM mAb to GXM. The pellicles from Hc GFP + Cn H99 displayed a slight, but significant increase in matrix reactivity to GXM-binding mAb compared to biofilms formed by Cn H99 alone. For both A and B, bars represent mean ± standard error of quadruplicates. (c) Cn glycan transfer to Hc surface is temperature dependent. More glycan transfer occurs at 37 °C compared to 30 °C during co-cultivation of Hc (GFP strain, FL1-H+) +Cn H99 in HAM’s F-12 medium as determined by flow cytometry using GXM-binding mAb 2D10 (IgGM) and a goat anti-mouse IgM-APC (FL4-H+), in comparison to controls or monospecies mixed Hc GFP + Cn H99 yeasts just before incubations with mAb, which displayed no PS transfer.