Ferrous iron uptake in Cryptococcus neoformans

Previous studies have implicated ferric reduction in the iron uptake pathway of the opportunistic pathogen Cryptococcus neoformans. Here we studied iron uptake directly, using 55Fe in the presence of reductants. Uptake was linear with respect to time and number of yeast cells. The plot of uptake versus concentration exhibited a steep rise up to about 1 microM, a plateau between 1 and 25 microM, and a second steep rise above 25 microM, consistent with high- and low-affinity uptake systems. A Km for high-affinity uptake was estimated to be 0.6 microM Fe(II); 1 microM was used for standardized uptake assays. At this concentration, the uptake rate was 110 +/- 3 pmol/10(6) cells/h. Iron repletion (15 microM) and copper starvation drastically decreased high-affinity iron uptake. Incubation at 0 degreesC or in the presence of 2 mM KCN abolished high-affinity iron uptake, suggesting that uptake requires metabolic energy. When exogenous reducing agents were not supplied and the culture was washed free of secreted reductants, uptake was reduced by 46%; the remaining uptake activity presumably was dependent upon the cell membrane ferric reductase. Further decreases in free Fe(II) levels achieved by trapping with bathophenanthroline disulfonate or reoxidizing with potassium nitrosodisulfonate reduced iron uptake very drastically, suggesting that it is the Fe(II) species which is transported by the high-affinity transporter. The uptake of Fe was stimulated two- to threefold by deferoxamine, but this increment could be abolished by copper starvation or inhibition of the ferric reductase by Pt, indicating that Fe solubilized by this molecule also entered the reductive iron uptake pathway.