Identifying a NORE involved in nitrosative stress response establishes a foundation for determining a mechanistic pathway of NO sensing in C. albicans. The 12-bp sequence TATTACCGTCGG, located about −230 bp from the YHB1 translation start site, is presently the only known cis-acting element involved in NO regulation in C. albicans. Mass spectrometry analysis identified five putative transcription factors that may have had specific affinity for the NORE sequence in vitro.
Several findings show that one of these five factors, CTA4, is required for NO signaling in C. albicans. When the five putative transcription factor genes were separately deleted, only the cta4Δ/cta4Δ homozygous deletion mutant manifested a decline in resistance to nitrosative stress. Exposing cta4Δ/cta4Δ cells to various NO sources decreased the growth rate and drastically reduced induction of YHB1 gene expression. ChIP results also indicated that the Cta4p protein localizes at the YHB1 regulatory region in vivo.
Data on CTA4
outside of this work are sparse. CTA4
was first isolated in a one-hybrid assay in S. cerevisiae
during a screen for a C. albicans trans
-activating regulator of pyruvate decarboxylase gene expression (21
). Later, various microarray studies reported that transcription of CTA4
is upregulated by exposure to nitrosative stress (19
) or growth in whole human blood (14
) and downregulated upon adherence to polystyrene (30
). The closest homolog for Cta4p in S. cerevisiae
is Oaf1p, an oleate receptor which heterodimerizes with Pip2p to activate peroxisomal β-oxidation genes for breaking down fatty acids (4
), but the C. albicans cta4
Δ mutant is able to utilize oleic acid as the sole carbon source, indicating that this gene does not regulate β-oxidation (M. Ramirez and M. Lorenz, unpublished observations). The next closest homolog among S. cerevisiae
proteins is Hap1p, a heme-responsive factor which binds DNA as a homodimer and is involved in oxygen regulation (52
Cta4p belongs in the Zn(II)2-Cys6 transcription factor family, a group of proteins unique to fungi, whose members bind DNA by means of a binuclear cluster of six cysteine residues that coordinate two zinc atoms. The C. albicans
genome is predicted to have genes for 77 of these factors (7
). The prototype for this class of proteins is the well-studied transcription factor Gal4p in S. cerevisiae
. The protein structure typically contains three domains: a DNA-binding domain almost always located at the N terminus, a central region believed to regulate the transcriptional activation activity of the factor, and an acidic activation domain at the C terminus (29
). Zn(II)2-Cys6 transcription factors function as homodimers, heterodimers, or sometimes monomers. Their cognate DNA binding sites often have CGG pairs that may be oriented as direct, inverted, or everted repeats. The spacing between these repeats varies widely between different proteins and is often a critical factor in recognition by a specific transcription factor (47
). The NORE sequence TATTACCGTCGG fits this archetype in that it contains an everted CGG repeat, but it also has the additional palindromic sequence TATTA. It may be comparable to Hap1p binding sites in S. cerevisiae
, which have similar, conserved TA-rich sequences that interact with the N terminus of Hap1p between the CGG direct repeats (23
Along with Cta4p, four other putative transcription factors from this zinc binuclear cluster family demonstrated in vitro affinity to NORE-coated magnetic particles: Zcf29p, Zcf36p, War1p, and Stb5p. Presently, we have no further evidence that these four transcription factors are biologically relevant to NO regulation. Their binding to the NORE could merely be an artifact of the artificial binding conditions, or they may have redundant functions in NO regulation that are not apparent when only one gene is eliminated at a time. The functions of the ZCF29
genes, which are also the closest homologs of CTA4
in the C. albicans
genome, are currently unknown. C. albicans WAR1
is known to provide resistance to the weak acid sorbate, a phenotype similar to its homolog in S. cerevisiae
. Although the function of STB5
in C. albicans
has not been investigated, its homolog in haploid S. cerevisiae
(32.5% amino acid sequence identity) has a variety of functions, including roles in multidrug resistance (1
), oxidative stress response (26
), and low-temperature response (2
The process of upregulation of the flavohemoglobin gene YHB1
during nitrosative stress in C. albicans
appears to differ from that in S. cerevisiae
. Liu et al., who first established the NO resistance function of YHB1
in S. cerevisiae
, did not detect any increase in YHB1
enzymatic activity during nitrosative stress (27
). Additionally, we previously reported constitutively activated YHB1
expression in S. cerevisiae
, regardless of the presence of NO-generating compounds (50
). However, Sarver and DeRisi documented induction of S. cerevisiae YHB1
transcription in a different strain specifically during nitrosative stress by transcription factor Fzf1p, a C2H2 zinc finger factor (42
). The closest homolog of S. cerevisiae FZF1
in C. albicans
has 19.3% amino acid sequence identity, and it is not known whether it can carry out similar functions. We have found that the analogous NO response in C. albicans
requires a different factor, Cta4p.
Another difference between C. albicans and S. cerevisiae is the role of the sulfite transporter SSU1. This gene mediates NO resistance under specific environmental conditions in S. cerevisiae, notably in synthetic medium. We hypothesized that the C. albicans SSU1 may have a similar function and that the yhb1Δ ssu1Δ double mutant would have a more severe phenotype, both in the presence of NO in vitro and in vivo. However, SSU1 has no role in NO detoxification in C. albicans under the conditions tested here, though its induction by NO is at least partly CTA4 dependent. Why SSU1 is part of the NO regulon is not immediately apparent.
We also demonstrated that deletion of the CTA4
gene produces a statistically significant reduction in virulence in a mouse infection model, though the effect was small. Deleting the YHB1
gene produced a similar mild attenuation of virulence, but this was not statistically significant, possibly because of the smaller number of mice tested. These data, together with earlier reports (19
), indicate that increasing susceptibility to NO has a small effect on virulence in the mouse tail vein model of candidiasis.
The mildness of the virulence reduction could indicate that YHB1 and CTA4 aid C. albicans survival against other NO sources that are not tested by this experiment. Injecting C. albicans directly into the bloodstream may be an appropriate model for certain situations, such as infection by contaminated implants, catheters, or needles, but this delivery method circumvents several common infection routes. Since C. albicans most often resides as a commensal organism on the mucosal linings of the urogenital and digestive tracts, it usually must infiltrate these barriers first before it can reach the bloodstream and disseminate throughout the body.
The NO in these environments could affect how vital YHB1
are to the survival and spread of this pathogen inside a host organism. For example, gastric NO concentrations in the human small intestine following ingestion of dietary nitrates can rise to levels thousands of times higher than in tissues (51
). Mouse models in which C. albicans
is introduced orally may be more informative in testing this. Additionally, the major function of the CTA4
genes could be more subtle than direct opposition against the host immune system; they may instead be more relevant in providing C. albicans
a survival advantage against other microorganisms vying for the same territory. Using more than one infection model will be important in establishing when and where these genes are critical for NO defense.
Our work takes the initial steps to decipher a pathway for NO signaling in C. albicans. Since this species is a major cause of fungal infections in humans, learning how it evades normal immune defenses and exploits weakened ones is essential for developing new strategies for treating infection.