Evidence for crucial components of the toxicity of Hst 5 toward C. albicans
has resulted in the development of two mechanistic models that previously appeared to be mutually exclusive. One model is based on evidence that Hst 5 induces rapid release of intracellular ATP and K+
in a Trk1p-dependent manner so that ion imbalance and irreversible cell volume loss occur in a mode resembling hyperosmotic shock. A second model is based on observations that Hst 5 associates with mitochondria, inhibits respiration, and induces the formation of ROS to produce oxidative stress (15
). In order to differentiate between these two models, we used genomic expression profiling of C. albicans
treated with Hst 5 to examine the global responses of cells to this toxin within 1 h of treatment. Here, for the first time, we provide evidence that the transcriptional response of C. albicans
to Hst 5 is that of cells undergoing adaptation to ionic or osmotic changes mediated by HOG pathways. However, osmotic stress also induces cellular changes in redox metabolism that overlap with oxidative-stress responses—both having in common the involvement of Hog1. Thus, the Hog1 MAPK pathway for stress response to Hst 5 suggests a common mechanism for unification of these two models.
The marked increase in the transcription of C. albicans
cell stress genes such as YHB1
, and HSP90
(Table ) following Hst 5 treatment that we observed initially suggested that cell response occurred via activation of one or more MAPK cascades. In order to delineate the role of each MAPK pathway in Hst 5-induced stress responses, we examined the susceptibilities of three C. albicans
strains, each with a mutation in the MKC1
, or HOG1
gene. Only Hog1-deleted cells exhibited hypersensitivity to Hst 5 peptide, suggesting that Hog1 is the predominant MAPK responsive to Hst 5-induced stresses. Since Hog1 MAPK is induced by both osmotic and oxidative (as well as heavy-metal) stresses in Candida
), we selected representative groups of osmotic- and oxidative-stress response genes for further quantitative analyses following 30 min, 60 min, and 120 min of Hst 5 exposure. Hst 5 induced significant increases in the expression of two groups of genes in a Hog1-dependent manner following 30 min and 60 min of treatment: general stress genes (HSP70
, and CTA1
) and genes involved in glycerol synthesis (RHR2
Osmotic stress induces profound adjustments of cellular metabolism to cope with shifts in cell turgor and ion balance, including production of glycerol, which serves as a cellular osmolyte. The behavior of cells following Hst 5 treatment is very similar to this classical biochemical response to hyperosmotic stress. We observed strong induction of gene transcripts (RHR2, GND1, and GPM1) encoding enzymes of the glycolytic pathway as well as Hog 1 phosphorylation following Hst 5 treatment. Also, intracellular glycerol accumulated in a dose-dependent manner following treatment with Hst 5. At lower Hst 5 doses, cells produced larger amounts of glycerol in proportion to the Hst 5 concentration; however, at higher Hst 5 doses, glycerol accumulation was found to be inversely proportional to the Hst 5 concentration. This bell curve response may be a result of increased cellular permeability accompanying increased Hst 5 doses. Hst 5 toxicity is characterized by rapid efflux of ions and ATP from cells, which may deplete cellular energy reserves and shut down metabolic pathways needed for new glycerol synthesis. Another possibility is that Hst 5 causes unregulated ion channels through which outflow of ions and ATP occurs, and these channels may also allow the passage of glycerol, resulting in a net loss of intracellular glycerol.
Interestingly, this classical osmotic-stress response may also explain why cells grown under anaerobic conditions are more resistant to Hst 5, since glycerol accumulation is accelerated under anaerobic conditions and results in cells capable of more-rapid osmotic adaptation (21
). Thus, anaerobic growth conditions or selective pressures that force cells into anaerobic growth (such as mitochondrial mutants) are preconditioned for rapid glycerol production and adaptation to the osmotic stresses produced by Hst 5. In this regard, we found that prestressing cells with sorbitol before Hst 5 treatment reduced peptide toxicity.
Osmotic stress also strongly diminishes the uptake of many amino acids, an effect resembling an amino acid starvation response. Indeed, our microarray experiments showed that a preponderance of genes down-regulated upon Hst 5 treatment were plasma membrane transporters, including GAP2 and HIP1 (general amino acid permeases), CDR11 (an ABC transporter), and NAG4 (encoding a monosaccharide transporter), suggesting energy preservation-like processes in cells treated with Hst 5. Since the cell wall plays a crucial role in maintaining cell turgor pressure, hyperosmotic stress induces cell wall remodeling and biogenesis through the HOG pathway in order to regulate cell osmolarity. In agreement with this model, we found that PIR1, encoding a Hog1-induced cell wall glycoprotein, was highly up-regulated by Hst 5 treatment.
Oxidative stresses may be generated as secondary effects of osmotic stress—for example, osmotic stress may interfere with the electron transport chain and enhance the production of ROS—or directly, due to oxidative damage and/or stimulation of redox metabolism. Hst 5 treatment caused increases in transcript levels of both Hog1-mediated (AHP1
, and TRX1
) and Hog1-independent (CAP1
) oxidative-stress genes, suggesting that both components could be involved. However, both cap1
cells and ssk1/ssk1
cells exhibited increased sensitivity to Hst 5, but only at higher doses at which osmotic-stress responses such as glycerol production are diminished. Thus, these results suggested that oxidative stress is generated secondarily to Hst 5 cell toxicity in both HOG-dependent and -independent pathways. In addition to genes encoding oxidoreductase enzymes involved in the metabolism of oxidized proteins (TRX1
and nitric oxide dioxygenase [YHB1
]), increased transcript levels for MET1
, involved in the biosynthesis of the oxidation-sensitive amino acid methionine, were observed, suggesting changes in cellular antioxidant capacity upon Hst 5 exposure. Interestingly, MET1
gene expression is induced in response to Hog1 activation (13
), since Hog1 regulates the function of genes involved in sulfur, methionine, alcohol, and lipid metabolism, as well as glycolysis (13
To further examine the role of oxidative and osmotic stresses in Hst 5 cytotoxicity, wild-type cells were treated with Hst 5 and grown under osmotic (sorbitol)- or oxidative (H2O2)-stress conditions. Osmotic stress substantially increased cell sensitivity to Hst 5, while oxidative stress had no effect on Hst 5 toxicity. Such facilitating interactions between the effects of sorbitol and Hst 5 further suggest a common underlying cellular response to osmotic stress and to Hst 5 toxin. Furthermore, our finding that oxidative stress did not enhance Hst 5 killing supports the view that oxidative stress is generated secondarily and is not an early or primary effect of Hst 5.
Here we report for the first time that the C. albicans
response to Hst 5 involves activation of the HOG pathway. Recently it was shown that treatment of Saccharomyces cerevisiae
with Pichia membranifaciens
killer toxin resulted in a coordinated transcriptional response, primarily through the HOG pathway, that was related to changes in ionic homeostasis (35
). Similarly, exposure of S. cerevisiae
cells to bacterial endotoxin induced phosphorylation of Hog1 (27
). Thus, the HOG pathway may be of significance in its ability to respond to changes in cellular osmolarity induced by a variety of toxins (as well as environmental conditions) in unicellular eukaryotes. In C. albicans
, the Hog1-mediated response to Hst 5 is both time and concentration dependent. HOG pathway responses are most protective against lower Hst 5 doses, since longer incubation times and higher Hst 5 doses result in the inability of cells to mount a full protective response. However, in the native environment of the oral cavity, where Hst 5 levels are often transient with respect to colonized Candida
cells and Hst concentrations are below 30 μM, the Hog1 response is likely to be a significant and effective challenge to physiological levels of Hst 5. Furthermore, adaptive activation of this pathway by cells subjected to prolonged or cyclical Hst 5 stresses may be a mechanism for the development of resistance to toxic cationic peptides. Thus, consideration of means to inactivate the HOG pathway in conjunction with doses of therapeutic peptides would enhance the efficacy of killing by Hst 5 and related natural antifungal agents. Upstream effectors of the HOG pathway, such as the osmosensor Sho1 or Sln1, are appealing candidates for this approach.