Figure 5

Genes regulated by ZAP1 are required for Sap6 induced autoaggregation and cell surface binding. (A) Geminated C. albicans (WT CAI4 and deletion mutants) were incubated with rSap6 (10 µM) for 15 min and average aggregate size was determined from 20 independent fields at 10X magnification using ImageJ software. None of the C. albicans ALS deletion mutants had aggregation defects (black bars), showing that Als proteins are not involved in autoaggregation. The C. albicans ZAP1 deletion mutant had the largest aggregation defect (aggregates were reduced by 90%) whereas ZAP1 complemention reversed the aggregation defect. Several other zinc acquisition and homeostasis mutants (PRA1, ZRT1, SOD6) showed aggregation reduced by more than 50%. Most of the genes whose deletion resulted in a significant (***P < 0.01) decrease in aggregate size are regulated by ZAP1 (hatched bars), although some (grey bars) are not known to be ZAP1 regulated. Mean ± SD of three independent experiments for each strain are shown. (B) C.albicans CAI4, Δzap1, Δzrt1 and Δpra1 deletion mutants, Δzap1/ZAP1, Δzrt1/ZRT1 and Δpra1/PRA1 complementation strains and ZAP1O/E, ZRT1O/E and PRA1O/E over-expressing strains were germinated and incubated with FITC-labeled rSap6 (F-Sap6) for 1 h. After washing, cell surface binding of F-rSap6 was documented microscopically at 63X. Cell surface binding of F-Sap6 to Δzap1, Δzrt1 and Δpra1 deletion strains was reduced compared with wild-type cells, while overexpression strains ZAP1O/E, ZRT1O/E and PRA1O/E showed increased cell surface binding of F-Sap6. Left panel, bright field; Center panel, FITC; and Right panel, merged bright field and FITC images.