Inducible defense mechanism against nitric oxide in Candida albicans

Abstract

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.

FIG. 1.

C. albicans contains three predicted flavohemoglobins with similarity to flavohemoglobins from other microbes. (A) Alignment of flavohemoglobins from several microbial genes. CaYhb1, -4, and -5 are the three predicted flavohemoglobins from C. albicans. CnYhb is the flavohemoglobin from Candida norvegensis (34), also known as Pichia norvegensis. ScYhb1 is from Saccharomyces cerevisiae, EcHmp is from Escherichia coli, and AeFhb is from Alcaligenes eutrophus. Amino acids important for binding the coenzymes heme and FAD in the X-ray structure of EcHmp (31) and AeFHb (16) are shown above the aligned sequences. Based on the alignment with the E. coli Hmp sequence (31), the globin domain comprises about the first third of the polypeptide chain, the FAD domain starts after the large arrow, and the NAD(P) domain is after the small arrowhead. Alignments were created with ClustalX and displayed with GeneDoc. (B) Phylogenetic tree of a subset of the microbial flavohemoglobins. The tree is based on an alignment (A) of the sum of the amino acids in these flavohemoglobins and is unrooted. Numbers adjoining the branches indicate bootstrap values (percentages from 500 replicates) based on results of the unweighted pair group method with arithmetic mean (40) calculated by using the PHYLIP program suite (J. Felsenstein; http://evolution.genetics.washington.edu/phylip.html). The different flavohemoglobins are shown together with the percent identity between select pairs.

FIG. 2.

Flavohemoglobin gene YHB1 is required for inducible NO metabolism in yeast. (A) NO consumption in yeast exposed either to air only (open bars) or to 960 ppm of gaseous NO in an air mixture (solid bars) for 60 min. (B) NOD activities in naïve and NO-exposed cells. Cells were harvested, extracts were prepared, and NOD activities were measured as described in Materials and Methods. scyhb1Δ indicates strain 15887, and cayhbxΔ/Δ refers to cayhbxΔ::HIS1/cayhbxΔ::dpl200 (Table 1). One milliunit is equivalent to 1 nmol per min. Error bars represent the standard deviation of three independent trials.

FIG. 3.

CaYHB1 mRNA is induced by NO in C. albicans. (A) CaYHB1 is induced by exposure to 960 ppm of NO gas (≤2 μM in solution). Naïve and NO-exposed cultures were grown as described in Materials and Methods, and cells were harvested at 15, 30, and 60 min, followed by mRNA isolation. (B) Induction of CaYHB1 mRNA by the NO-releasing compound NOC-18. A total of 3 mM NOC-18 was added to exponentially growing yeast (37°C), and cells were collected at 0, 15, 30, and 60 min for RNA extraction. (C) Induction of YHB1 in C. albicans and S. cerevisiae by nitrite. Various concentrations of sodium nitrite (NaNO₂) were added to log-phase cultures of the S. cerevisiae strain BY4742 (grown at 30°C) or the C. albicans strain RM1000 (grown at 37°C). After 30 min of treatment, cells were collected and mRNA was extracted. (D) A total of 25 mM NaNO₂ was added to exponential cultures of BY4742 (30°C) or RM1000 (37°C), and after 0, 5, 15, 30, and 60 min, cells were harvested for mRNA isolation. ACT1 was probed as a loading control for Saccharomyces RNA; CaTEF1 was the control for Candida samples. (E) Induction of CaYHB1-lacZ by nitrite. A C. albicans strain containing a CaYHB1-lacZ reporter gene plasmid, integrated into a chromosome downstream of the HWP1 gene (see Materials and Methods), was grown in rich YEPD medium with or without 10 mM nitrite at 37°C for 1 h, and β-galactosidase activity in cell extracts was measured as described previously (2).

FIG. 4.

Deletion of CaYHB1 increases sensitivity to NO-generating compounds and to nitrite. Growth of wild-type (RM1000; solid circles), cayhb1Δ/cayhb1Δ (open circles), cayhb4Δ/cayhb4Δ (solid triangles), or cayhb5Δ/cayhb5Δ (open triangles) yeast cultures was measured following treatment of log-phase cultures with control solvent or NOC-18 (A), S-nitrosoglutathione (GSNO) (B), or sodium nitrite (NaNO₂) (C) at the indicated concentrations. After 4 h of treatment, growth was calculated from the change in turbidity at 600 nm (A₆₀₀^{t = 4} −A₆₀₀^{t = 0}). Data are from representative experiments that were repeated at least two times with similar results.

FIG. 5.

Reinsertion of CaYHB1 complements sensitivity of the cayhb1Δ/cayhb1Δ strain to sodium nitrite. Growth of cultures of the wild-type strain (RM1000; solid circles) or mutant strains of the types cayhb1Δ::HIS1/cayhb1Δ::dpl200 (open triangles), cayhb1Δ::dpl200/CaYHB1 (solid squares), cayhb1Δ::HIS1/cayhb1Δ::dpl200 transformed with empty plasmid pABSKII (open diamonds), or cayhb1Δ::HIS1/cayhb1Δ::dpl200 transformed with complementation plasmid pABSKII CaYHB1 (solid triangles) was determined following treatment with the indicated concentrations of sodium nitrite for 4 h. Growth was determined from the change in turbidity at 600 nm. The RM1000, cayhb1Δ/Δ, and cayhb1Δ/+ strains were each grown in YEPD medium supplemented with uridine, while the strains containing plasmid pABSKII or pABSKII CaYHB1 were grown in YEPD medium without uridine to maintain selection for the URA3-containing plasmid. The exact reason for slower growth of the latter cultures is not known, but it may reflect a low activity of the plasmid URA3 gene. Results are representative of two or more experiments.

FIG. 6.

C. albicans strains lacking CaYHB1 show attenuated virulence. The His⁺ Ura⁺ C. albicans strains used were the CAF2, Δyhb1 (yhb1Δ/yhb1Δ ura3Δ/URA3^c), and YHB1 reconstituted (yhb1Δ/YHB1^c ura3Δ/URA3^c) strains shown. Tail vein injection with Candida was performed on day 0.