Mammalian heme oxygenases play important roles in immune regulation by producing immunosuppressive CO. The pathogenic yeast C. albicans encodes a heme oxygenase, Hmx1, that is specifically induced by the host protein hemoglobin, suggesting a role in the pathogenesis of disseminated bloodstream infections. We show that exposing mice to therapeutic levels of CO increases C. albicans virulence, whereas a HMX1 null strain has decreased virulence in murine disseminated candidiasis. Levels of several regulatory cytokines and chemokines are decreased in mice infected with the null strain, and initial lesions in the kidney are more rapidly cleared following PMN infiltration. Reconstitution of one or both alleles restores virulence to the level of wild type. Growth in vitro and initial organ burdens in infected mice are not decreased and host iron overload does not restore virulence for the null strain, suggesting that early growth in the host is not limited by Hmx1-mediated iron scavenging. In contrast, inhaled CO partially reverses the virulence defect of the null strain and restores several host cytokine responses to wild type levels. Collectively, these results show that C. albicans Hmx1 expression and CO production limit the host immune response and contribute to the pathogenesis of candidemia. Hemiascomycetes, including the pathogen C. albicans, acquire nitrogen from urea using the urea amidolyase Dur1,2, whereas all other higher fungi use primarily the nickel-containing urease. Urea metabolism via Dur1,2 is important for resistance to innate host immunity in C. albicans infections. To further characterize urea metabolism in C. albicans we examined the function of seven putative urea transporters. Gene disruption established that Dur3, encoded by orf 19.781, is the predominant transporter. [(14)C]Urea uptake was energy-dependent and decreased approximately sevenfold in a dur3 deletion mutant. DUR1,2 and DUR3 expression was strongly induced by urea, whereas the other putative transporter genes were induced less than twofold. Immediate induction of DUR3 by urea was independent of its metabolism via Dur1,2, but further slow induction of DUR3 required the Dur1,2 pathway. We investigated the role of the GATA transcription factors Gat1 and Gln3 in DUR1,2 and DUR3 expression. Urea induction of DUR1,2 was reduced in a gat1 deletion mutant, strongly reduced in a gln3 deletion mutant, and abolished in a gat/gln3 double deletion mutant. In contrast, DUR3 induction by urea was preserved in both single mutants but reduced in the double mutant, suggesting that additional signalling mechanisms regulate DUR3 expression. These results establish Dur3 as the major urea transporter in C. albicans and provide additional insights into the control of urea utilization by this pathogen. HBR1 (hemoglobin response gene 1) is an essential gene in C. albicans that positively regulates mating type locus MTL-alpha gene expression and thereby regulates cell type-specific developmental genes. Hbr1p contains a phosphate-binding loop (P-loop), a highly conserved motif characteristic of ATP- and GTP-binding proteins. Recombinant Hbr1p was isolated in an oligomeric state that specifically bound ATP with K(d) =2 micromolar. ATP but not ADP, AMP, GTP, or dATP specifically protected Hbr1p from proteolysis by trypsin. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and the less conserved glycine (G19S) decreased the binding affinity for soluble ATP and ATP immobilized through its gamma-phosphate. ATP bound somewhat more avidly than ATPgamma-S to wild type and mutant Hbr1p. Although Hbr1p exhibits sequence motifs characteristic of adenylate kinases, and adenylate kinase and ATPase activities have been reported for the apparent human ortholog of Hbr1p, assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative. Overexpression of wild type but not the mutant forms of Hbr1p restored MTl-alpha-a2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIASC009173-08
Application #
8350062
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2011
Total Cost
$428,036
Indirect Cost
Name
National Cancer Institute Division of Clinical Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Navarathna, Dhammika H M L P; Pathirana, Ruvini U; Lionakis, Michail S et al. (2016) Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis. PLoS One 11:e0164449
Navarathna, Dhammika H; Roberts, David D; Munasinghe, Jeeva et al. (2016) Imaging Candida Infections in the Host. Methods Mol Biol 1356:69-78
Navarathna, Dhammika H M L P; Stein, Erica V; Lessey-Morillon, Elizabeth C et al. (2015) CD47 Promotes Protective Innate and Adaptive Immunity in a Mouse Model of Disseminated Candidiasis. PLoS One 10:e0128220
Pendrak, Michael L; Roberts, David D (2015) Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions. PLoS One 10:e0126919
Navarathna, Dhammika H M L P; Munasinghe, Jeeva; Lizak, Martin J et al. (2013) MRI confirms loss of blood-brain barrier integrity in a mouse model of disseminated candidiasis. NMR Biomed 26:1125-34
Martin-Manso, Gema; Navarathna, Dhammika H M L P; Galli, Susana et al. (2012) Endogenous thrombospondin-1 regulates leukocyte recruitment and activation and accelerates death from systemic candidiasis. PLoS One 7:e48775
Navarathna, Dhammika H M L P; Lionakis, Michail S; Lizak, Martin J et al. (2012) Urea amidolyase (DUR1,2) contributes to virulence and kidney pathogenesis of Candida albicans. PLoS One 7:e48475
Navarathna, Dhammika H M L P; Das, Aditi; Morschhauser, Joachim et al. (2011) Dur3 is the major urea transporter in Candida albicans and is co-regulated with the urea amidolyase Dur1,2. Microbiology 157:270-9
Pendrak, Michael L; Roberts, David D (2011) Ribosomal RNA processing in Candida albicans. RNA 17:2235-48
Peterson, Alexander W; Pendrak, Michael L; Roberts, David D (2011) ATP binding to hemoglobin response gene 1 protein is necessary for regulation of the mating type locus in Candida albicans. J Biol Chem 286:13914-24

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