GLN3 encodes a global regulator of nitrogen metabolism and virulence of C. albicans

Abstract

The function of GLN3, a GATA factor encoding gene, in nitrogen metabolism of Candida albicans was examined. GLN3 null mutants had reduced growth rates on multiple nitrogen sources. More severe growth defects were observed in mutants lacking both GLN3 and GAT1, a second GATA factor gene. GLN3 was an activator of two genes involved in ammonium assimilation, GDH3, encoding NADP-dependent glutamate dehydrogenase, and MEP2, which encodes an ammonium permease. GAT1 contributed to MEP2 expression, but not that of GDH3. A putative general amino acid permease gene, GAP2, was also activated by both GLN3 and GAT1, but activation by GLN3 was nitrogen source dependent. GLN3 was constitutively expressed, but GAT1 expression varied with nitrogen source and was reduced 2- to 3-fold in gln3 mutants. Both gln3 and gat1 mutants exhibited reduced sensitivity to rapamycin, suggesting they function downstream of TOR kinase. Hyphae formation by gln3 and gat1 mutants differed in relation to nitrogen source. The gln3 mutants formed hyphae on several nitrogen sources, but not ammonium or urea, suggesting a defect in ammonium assimilation. Virulence of gln3 mutants was reduced in a murine model of disseminated disease. We conclude that GLN3 has a broad role in nitrogen metabolism, partially overlapping, but distinct from that of GAT1, and that its function is important for the ability of C. albicans to survive within the host environment.

Fig. 1. Sequence relatedness of CaGln3p

A ClustalX alignment (Jeanmougin et al., 1998) of CaGln3p and ScGln3p is shown in panel A. Identical residues are marked with an asterisk. The zinc-finger and carboxy-terminal domains are boxed. Panel B shows an alignment of the carboxy-termini of CaGln3p and GATA factors of other fungi; Sc, S. cerevisiae; Mg, Magnaporthe grisea Nut1 (Acc.# Q01168), An, A. nidulans Area (Acc.# CAA36731); Gf, Gibberella fujikuroi Area (Acc.# P78688); Cf, Cladosporium fulvum Nrf1 (Acc.# AAG48616); Nc, Neurospora crassa Nit-2 (Acc.# A34755). The thin lines enclose residues that are identical in Ca_Gln3p and Sc_Gln3p. The thick lines enclose amino acids conserved in >50% of the sequences shown.

Fig. 2. Effect of nitrogen source and GATA-factor mutations on gene expression

Northern blot analysis of RNA hybridized with GDH3 (A.), MEP2 (B. and C.), GAP2 (D.) or GAT1 (E.). RNA was extracted from strains CAI-12 (Parent), GUI-11 (Gln3⁻), the rescued gln3 mutant GLR-11 (Gln3⁺-R), CTL-7 (Gat1⁻), the rescued gat1 mutant CTL-5 (Gat1⁺-R), or GGU-11 (Gln3⁻ Gat1⁻) cultured with 10 mM proline (A., C., D., and E.) or glutamine (D.). RNA samples used in B. were prepared from strain CAI-12 cultured with 10 mM of the indicated nitrogen source. As a control, all blots were hybridized with HHT1.

Fig. 3. Effect of GATA-factor mutations on rapamycin sensitivity

Serial dilutions of strains CAI-12 (Parent), GUI-11 (Gln3⁻), the rescued gln3 mutant GLR-11 (Gln3⁺-R), CTL-7 (Gat1⁻), the rescued gat1 mutant CTL-5 (Gat1⁺-R), or GGU-11 (Gln3⁻ Gat1⁻) were spotted on YPD or YPD + 30 ng/ml rapamycin and imaged after incubation 5 days at 30°C.

Fig. 4. Filamentation phenotype of GATA-factor mutants

Strains CAI-12 (parental), GUI-11 (Gln3⁻), GLR-11 (Gln3⁺ rescued), CTL-7 (Gat1⁻), CTL-5 (Gat1⁺ rescued), or GGU-11 (Gln3⁻ Gat1⁻) were nitrogen starved and 2 μl of each, containing 1 × 10⁶ cells, was spotted on medium containing 1 mM of the indicated nitrogen source. Colonies were imaged after incubated 4 d at 37°C. A section of the colony from each strain is shown.

Fig. 5. Effect of nutrient conditions on hyphae formation by gln3 mutant

Strains CAI-12 (parental), GUI-11 (Gln3⁻) were nitrogen starved and 2 μl of each, containing 1 × 10⁶ cells, was spotted on medium containing the indicated concentration of ammonium and glucose. Colonies were imaged after incubated 4 d at 37°C. A section of the colony from each strain is shown.

Fig. 6. Virulence phenotype of gln3 mutants

Ten BALB/c mice were infected with 1 × 10⁶ cells of strain CAI-12 (GLN3/GLN3), a heterozygous mutant CWL-5A (gln3/GLN3), a null mutant, GUI-11 (gln3/gln3) or a rescued mutant, GLR-11 (GLN3/gln3) and monitored for 19 days post infection.