A major defense mechanism mounted against invading pathogens is the production of nitric oxide (.
NO), a free radical that rapidly diffuses across cell membranes and is capable of reacting with a variety of molecules and causing multiple types of cell damage. The relationship between .
NO and individual human pathogens is complex and has not been systematically investigated for C. albicans
. However, available evidence supports the view that .
NO is important for the control of C. albicans
infections. For example, it has been reported that mice deficient in .
NO production are hypersensitive to C. albicans
infections as judged by organ load (Netea et al., 2002
). In addition, killing of C. albicans
by murine saliva and macrophages has been shown, in some cases, to require NOS2
(Elahi et al., 2001
; Netea et al., 2002
; Balish et al., 2005
Although the physiological importance of .
NO in control of C. albicans
requires further investigation, it has been well established that .
NO production by the NOS2 enzyme is crucial for the control of other pathogens, including Mycobacterium tuberculosis, Leishmania
spp. (for review see Nathan and Shiloh, 2000
), and Cryptococcus neoformans
(de Jesus-Berrios et al., 2003
). In the latter organism, it was shown that deletion of a flavohemoglobin gene (FHB1
) results in attenuated virulence in wild-type mice that can be suppressed in infections of NOS2-/-
mice (de Jesus-Berrios et al., 2003
Because of the established importance of .
NO in the control of numerous other pathogens, we examined the genome-wide response of C. albicans
NO. In this article, we identified 131 genes that are induced or repressed in wild-type C. albicans
in response to .
NO (see , , and Supplementary Table 1). Based on their kinetic profiles, these genes fall into three classes: 1) transiently induced genes, 2) transiently repressed genes, and 3) persistently induced genes. As described in Results
, we propose that the transiently induced and repressed genes primarily encode proteins involved in counteracting the secondary .
NO-induced effects such as oxidative stress, whereas the persistently induced genes encode proteins that specifically protect against .
NO. Transiently induced transcripts encode oxidative stress proteins such as glutathione-conjugating and -modifying enzymes, NADPH oxidoreductases/dehydrogenases, catalase, iron acquisition proteins, transcription factors, sulfur assimilation enzymes, transporters of oligopeptides, drugs, and heavy metals, and heme-binding proteins (see Results
and ). Transiently repressed transcripts encode subunits of the mitochondrial electron transport chain and ribosomal proteins (see Results
and Supplementary Table 1). Only nine genes are persistently induced by .
NO; these include YHB1
, two alternative oxidases, two putative cell surface heme-binding proteins, putative transporters for copper, sulfite, and iron, and a conserved protein of unknown function (see ). As described in the accompanying article (Sarver and DeRisi, 2005
), two of these nine genes (YHB1
a putative sulfite transporter) are also persistently induced when S. cerevisiae
is exposed to .
On deletion of the YHB1
gene in C. albicans
, the genome-wide expression profile in response to .
NO is more pronounced. That is, the genes that show only transient induction or repression in wild-type strains show prolonged and enhanced changes in expression in the yhb1
Δ deletion strain (, EXPT 4). In addition, a cluster of 34 new genes showed significant induction only in the yhb1
Δ strain (, gray vertical bar); these genes encode proteins involved in the repair of DNA damage, as well as additional proteins involved in processes such as oxidative stress, iron acquisition, and transport (Supplementary Table 2). The prolonged and enhanced changes in .
NO-induced gene expression observed upon removal of the YHB1
gene, strongly supports the idea that Yhb1 is indeed functioning to detoxify .
NO in C. albicans
. Consistent with this idea, growth of the yhb1
Δ mutant in vitro is much more sensitive to .
NO than is wild type (). This latter result was also reported by Ullmann et al.
), using an independently derived yhb1
Also in agreement with Ullmann et al.
), we demonstrated that the yhb1
Δ strain is moderately attenuated for virulence in BALB/c mice, as assessed by the mouse tail vein model of disseminated candidiasis (). To our surprise, however, the C. albicans yhb1
Δ virulence defect was not suppressed in mice deleted for the NOS2
gene (). This result implies that the virulence defect of the yhb1
Δ mutant is not solely due to an inability to detoxify .
NO and implicates an additional function as being responsible. One possibility is a role for YHB1
in the control of filamentous growth. As described in the Results
section, the yhb1
Δ strain is hyperfilamentous, as observed as altered colony morphologies on laboratory media () and as the expression of “filament-specific” genes under conditions where they should be repressed (). Several C. albicans
hyperfilamentous mutants have been shown to have defects in virulence; these include tup1
Δ and nrg1
Δ (both of which are severely hyperfilamentous) (Braun et al.
; Murad et al., 2001
), as well as rfg1
Δ (which has a mild hyperfilamentous phenotype similar to that of yhb1
Δ; Kadosh and Johnson, 2001
). It is also possible that the yhb1
Δ strain has additional defects that render it less virulent. For example, YHB1
is also induced in media rich in iron (Lan et al., 2004
), upon phagocytosis by macrophages (Lorenz et al., 2004
), and by sodium sulfite (our unpublished result). Thus, Yhb1 likely has roles in addition to protecting against nitrosative stress, and at least one of these additional functions must be required for full virulence in the mouse tail vein model of candidiasis.
Another unanticipated result concerns the susceptibility of mice lacking the NOS2
gene to infection by C. albicans
. We found that NOS2-/-
mice are not significantly more susceptible than immunocompetent mice to killing by C. albicans
, as assessed by the tail vein model of infection (). These results indicate that NOS2 production of .
NO has little or no effect on the susceptibility of mice to C. albicans
infection through this route. By introducing C. albicans
directly into the venous system, this model bypasses, for example, mucosal and epithelial barriers, and thereby escapes a step where .
NO production may have a significant effect on the outcome of the infection. These observations suggest that the yhb1
Δ mutant strain could be a useful tool to identify models of infection that do require .
NO for containing infections caused by C. albicans
and, by implication, to identify the steps of infection at which .
NO is important for defense against this pathogen. Given the high conservation of the .
NO transcriptional response in C. albicans
and S. cerevisiae
(see accompanying article [Sarver and DeRisi, 2005
]), it seems likely that both fungi routinely encounter concentrated levels of .
NO in their environment. Further studies employing the C. albicans yhb1
Δ mutant strain should illuminate the host microenvironment where .
NO presents a serious threat for C. albicans