Previous studies on Δhog1 mutants from C. albicans and Cryptococcus neoformans showed that deletion of HOG1 led to the de-repression of several genes known to be upregulated under restricted iron conditions [27, 50]. In C. albicans, this group of genes included RBT5, FRE10, FTR1, FET34, orf19.251, PMH7, ECM331, CAT1, DDR48, YOR009 and HSP12[22, 23, 27].
Whether this phenotype was due to a direct involvement of Hog1p in the regulation of the iron responsive network or due to indirect effects, such as perturbations of copper metabolism, which may have impaired the functionality of iron uptake proteins was not yet studied.
As expected, high levels of extracellular iron increased the formation of intracellular ROS. Thus, we used intracellular ROS levels together with the removal of iron from growth medium as indicators of iron entry into the cells. We detected increased basal ROS levels in the Δhog1 mutants, as previously reported . These ROS levels were further increased by exposure to 30 μM Fe3+ confirming that iron was taken up by Δhog1 cells. Moreover, iron ions were removed from the growth medium with the same efficiency by Δhog1 as by the reference (DAY286) cells. Thus, Hog1p dependent phenotypes of the C. albicans response to iron were not due to iron uptake deficiencies, but could be rather due to the involvement of Hog1p in the response to iron availability. This is supported by our data on the transient hyper-phosphorylation of Hog1p during exposure of cells to high iron concentrations.
Elevated iron concentrations induced a flocculent phenotype of C. albicans, which was dependent on the presence of both Hog1p and Pbs2p, as well as on protein synthesis. As high iron concentrations led to increased phosphorylation of Hog1p, this could induce the synthesis of proteins of which some mediate cell aggregation. This iron triggered activation of Hog1p is likely not related to oxidative stress, as the potent radical scavenger NAC did not prevent the flocculent phenotype upon exposure to high iron concentrations, while it decreased intracellular ROS levels. For the closely related yeast S. cerevisiae, a function of ScHog1p in cell aggregation was reported, in that hyperactive ScHog1p mutants resulted in increased flocculation .
First hints on an involvement of Hog1p in the response of C. albicans to iron came from the observation of the de-repression of several iron uptake genes in the Δhog1 mutant under otherwise repressive conditions . In agreement with these gene expression data, we observed increased MCFOs protein levels and ferric reductase activity in Δhog1 mutants. Furthermore we found that MCFOs were also de-repressed in Δpbs2 mutants, indicating that the HOG1 mediated regulation of MCFOs was dependent on PBS2. Remarkably, induction of these components in RIM was not strictly dependent on Hog1p, as this induction was also observed in the Δhog1 mutant. Thus deletion of HOG1 de-repressed components of the iron uptake system, and this elevated basal level was further enhanced when iron availability was limited.
Hog1p was shown to be essential for C. albicans under oxidative stress conditions . Our data indicated that the absence of HOG1 reduced the metabolic activity of the cells after exposure to high iron concentrations compared to wild type cells. Taking in account that exposure of Δhog1 cells to high iron concentrations further increased the comparably high basal intracellular ROS levels in the mutant, the decreased viability of the Δhog1 mutant under such conditions could be due to elevated oxidative stress. However, other mechanisms independent from Hog1p were also described for the initiation of oxidative stress responses . These mechanisms could allow also the mutant strains to adapt to the stress conditions so that the reduced viability was observed only as immediate response and did not lead to significant growth defects.
It has yet to be elucidated which elements downstream of Hog1p provide the link between the HOG pathway and factors which regulate reductive iron uptake. As many Hog1p repressed genes, including those involved in iron uptake (FET34, FRE10, FTR1 and RBT5), were also found to be repressed by Tup1p , a role for this global co-repressor downstream of Hog1p could be assumed. Indeed, a role of Tup1p in regulating iron uptake has been reported . However, the details remain to be elucidated.
In this study, we used several single gene deletion mutants which were generated by different approaches [31, 44, 53, 54]. All mutant strains were descendants of the strain CAI-4 , in which both copies of IRO1 are deleted. Additionally, all strains ectopically express URA3.
IRO1 is a gene that encodes a transcription factor with a potential role in iron utilization. Expression of IRO1 in a Δaft1 S. cerevisiae strain restored growth in iron depleted media. However, a role of IRO1 in C. albicans iron metabolism is not confirmed . On the other hand, ectopic expression of URA3 has been shown to affect several features of C. albicans, such as hyphal morphology, adhesion, virulence and cellular proteome in addition to Ura3p activity [57, 58].
In all of our experiments, the DAY286 reference strain behaved similar to the WT SC5314. Additionally, CNC13 and JMR114 (Δhog1) as well as BRD3 and JJH31 (Δpbs2) showed similar features. Thus, no effects of the ectopic expression of URA3 or the absence of IRO1 were observed.