Fig 12. Susceptibility of fre8Δ/Δ cells to killing by neutrophils and macrophages.

(A) SC5314 WT or the isogenic fre8Δ/Δ mutant biofilms were grown for 24 hours in 96 well plates and the relative fungal burden was estimated using the XTT assay as described in Materials and Methods. Results were normalized to SC5314 biofilm allowing for averaging of 3 independent experiments. (B) 24 hour SC5314 or fre8Δ/Δ biofilms were incubated with or without human neutrophils (E:T ratio 1:2) for 4 hours and an XTT metabolic assay was used to estimate fungal burden. No biofilm controls were included to estimate neutrophil contribution to XTT assays. Neutrophil contributions were subtracted from biofilm XTT assays to calculate fungal inhibition. Results represent the averages of 3 independent experimental trials. The increase in inhibition of fre8Δ/Δ cells by neutrophils was statistically significant (P ≤ 0.04 by T-test). (C) WT SC5314 or the isogenic fre8Δ/Δ, sod5Δ/Δ or the FRE8 complemented fre8Δ/Δ (fre8Δ/Δ Res) were tested for killing by BMDM as described in Materials and Methods. Results represent the averages of 9 samples over two experimental trials. The increased killing of sod5Δ/Δ and fre8Δ/Δ cells compared to WT or to the fre8Δ/Δ Res was statistically significant ****p<0.0001 by one-way ANOVA with Tukey post-test. There was no statistically significant difference between WT and fre8Δ/Δ Res, while the increased killing in sod5Δ/Δ compared to fre8Δ/Δ was significant (p = 0.004) by one-way ANOVA with Tukey post-test.