Transcriptional response of Candida albicans to nitric oxide and the role of the YHB1 gene in nitrosative stress and virulence

Abstract

Here, we investigate how Candida albicans, the most prevalent human fungal pathogen, protects itself from nitric oxide (*NO), an antimicrobial compound produced by the innate immune system. We show that exposure of C. albicans to *NO elicits a reproducible and specific transcriptional response as determined by genome-wide microarray analysis. Many genes are transiently induced or repressed by *NO, whereas a set of nine genes remain at elevated levels during *NO exposure. The most highly induced gene in this latter category is YHB1, a flavohemoglobin that detoxifies *NO in C. albicans and other microbes. We show that C. albicans strains deleted for YHB1 have two phenotypes in vitro; they are hypersensitive to *NO and they are hyperfilamentous. In a mouse model of disseminated candidiasis, a YHB1 deleted C. albicans strain shows moderately attenuated virulence, but the virulence defect is not suppressed by deletion of the host NOS2 gene. These results suggest that *NO production is not a prime determinant of virulence in the mouse tail vein model of candidiasis and that the attenuated virulence of a yhb1delta/yhb1delta strain is attributable to a defect other than its reduced ability to detoxify *NO.

Figure 1.

Genome-wide expression profile of C. albicans in response to nitric oxide (^.NO). Four independent time courses were analyzed (EXPT 1-4) in CAF2-1, RM1, RM1000, and in a yhb1Δ/yhb1Δ mutant strain (MMY272; see Table 1 for strain details). Samples were collected 10, 40, 70, and 120 min (triangles represent increasing time) after exposure to 1.0 mM DPTA NONOate (treated) or 13.3 nM NaOH (mock-treated), and all data were transformed over an untreated zero time-point sample (black column at the beginning of each experiment). Red and green squares indicate induction and repression, respectively, black indicates no change in expression, and gray indicates no data available. (A) Sixty-five genes are up-regulated in response to ^.NO. Nine genes (yellow vertical bars) are persistently up-regulated throughout the 2-h time course in all strain backgrounds (EXPT 1-4). Fifty-six genes (blue vertical bar) are transiently up-regulated in YHB1 intact strains (EXPT 1-3) and show prolonged induction in a yhb1Δ/yhb1Δ mutant strain (EXPT 4). An additional 34 genes (gray vertical bar) are only up-regulated in a yhb1Δ/yhb1Δ mutant strain (EXPT 4). (B) Sixty-five genes are down-regulated in response to ^.NO (orange vertical bar). In YHB1 intact strains, these genes are transiently down-regulated at the 10-min time point and return to baseline expression levels by the 40-min time point (EXPT 1-3). In a yhb1Δ/yhb1Δ mutant strain, the majority of these genes are persistently down-regulated throughout the 2-h time course (EXPT 4).

Figure 2.

Fold induction of C. albicans flavohemoglobin genes (YHB1, YHB4, YHB5) in response to nitric oxide (^.NO). The PAT1 transcript was used as a control and reveals no induction by ^.NO. Samples were collected 10 (yellow) and 180 (blue) min after exposure to 1.0 mM DPTA NONOate, 8.0 mM Carboxy PTIO (Scavenger), or a combination of both chemicals. Addition of a chemical is indicated by a (+) and no-addition indicated by a (-). Three independent quantitative RT-PCR reactions were performed, and in each experiment, the signals were divided by an untreated zero time-point sample. The plot and error bars represent the average of the three experiments.

Figure 3.

The yhb1Δ/yhb1Δ mutant is hypersensitive to nitric oxide (^.NO) in vitro. C. albicans cultures were exposed to five different concentrations of DPTA NONOate (0.0, 0.5, 1.0, 2.0, 3.5 mM) and after 8 h of exposure, were serial diluted and plated. After 24 h of incubation, colony-forming units were counted. Wild-type (BH117), yhb1Δ/YHB1 heterozygous (BH115), and yhb1Δ/yhb1::YHB1 addback (BH94, BH96, BH98) strains showed comparable survival at low concentrations of DPTA NONOate (0.5 and 1.0 mM); the yhb1Δ/yhb1Δ mutant (BH79, •), however, is hypersensitive to ^.NO at these concentrations. At higher concentrations of DPTA NONOate (3.5 mM), all strains are sensitive.

Figure 4.

The yhb1Δ/yhb1Δ mutant is hyperfilamentous under nonfilamenting conditions. Wild-type (BH117), yhb1Δ/YHB1 heterozygous (BH115), yhb1Δ/yhb1Δ mutant (BH79), and yhb1Δ/yhb1::YHB1 addback (BH98) strains were tested for filamentation defects on YEPD at 37°C.

Figure 5.

Hyphal-specific genes are induced in the yhb1Δ/yhb1Δ mutant in YEPD at 37° in the absence of nitric oxide (^.NO). Genes induced over sixfold in the yhb1Δ/yhb1Δ mutant (BH79) are shown (mutant (BH79) transformed over wild type (BH117)). The induction of hyphal-specific transcripts supports the hyperfilamentous plate phenotype of the yhb1Δ/yhb1Δ mutant (see Figure 4).

Figure 6.

YHB1 is important for virulence in a mouse tail vein model of systemic candidiasis. (A) Groups of 10 immunocompetent BALB/c mice were tail vein injected with 3 × 10⁵ cells of wild-type (BH117, yellow squares), yhb1Δ/yhb1Δ mutant (BH79, pink squares), or yhb1Δ/yhb1::YHB1 addback (BH98, blue squares) strains, and survival was monitored. (B) Groups of 10 or 11 congenic C57BL/6 NOS2^+/+ or NOS2^-/- mice were tail vein injected with 5 × 10⁵ cells of wild-type (BH117, yellow squares and green triangles) or yhb1Δ/yhb1Δ mutant (BH79, pink squares and gray triangles) strains, and survival was monitored.