Fungal homoserine kinase (thr1Delta) mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation

Abstract

The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae homoserine kinase (thr1Delta) and threonine synthase (thr4Delta) mutants. In striking contrast to aspartate kinase (hom3Delta) mutants, S. cerevisiae thr1Delta and thr4Delta mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1Delta mutants, but not hom3Delta mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1Delta and thr4Delta mutants as well as C. albicans thr1Delta mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1Delta mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.

Fig. 1.

Survival of S. cerevisiae thr1Δ and thr4Δ strains in vivo. Results were the average of experiments with two independent mutants with the same gene deleted. hom3Δ mutant results were previously published (31) and were included as a comparison. CI values = (experimental strain/reference strain)_{t = x}/(experimental strain/reference strain)_{t = 0}.

Fig. 2.

Virulence phenotypes of C. albicans threonine pathway mutants.

Fig. 3.

(A) Serum sensitivity of S. cerevisiae and C. albicans threonine pathway mutants and the S. cerevisiae thr1Δ gcn4Δ mutant. Strains were incubated in serum for the time designated, and 5-μl volumes of 10-fold dilutions were plated on YPD medium. (B) Time course for serum killing of S. cerevisiae thr1Δ mutants.

Fig. 4.

Effect of pH, calcium chelation, and threonine addition on serum sensitivity of S. cerevisiae and C. albicans thr1Δ mutants. Strains were incubated in serum for the time designated, and 5-μl volumes of 10-fold dilutions were plated onto YPD.

Fig. 5.

Threonine starvation is cytocidal for S. cerevisiae and C. albicans thr1Δ mutants. Strains were incubated in SD + Met or SD + Met + low Thr (39 μg ml⁻¹; equivalent to serum levels), and at the designated times, aliquots were removed and dilutions plated to YPD.