Identification of hexose transporter-like sensor HXS1 and functional hexose transporter HXT1 in the methylotrophic yeast Hansenula polymorpha.

We identified in the methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) a novel hexose transporter homologue gene, HXS1 (hexose sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene, HXT1 (hexose transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the Saccharomyces cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1Delta deletion strain. Our combined results suggest that in the Crabtree-negative yeast H. polymorpha, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.

Combined inactivation of the Candida albicans GPR1 and TPS2 genes results in avirulence in a mouse model for systemic infection.

Inhibition of the biosynthesis of trehalose, a well-known stress protectant in pathogens, is an interesting approach for antifungal or antibacterial therapy. Deletion of TPS2, encoding trehalose-6-phosphate (T6P) phosphatase, results in strongly reduced virulence of Candida albicans due to accumulation of T6P instead of trehalose in response to stress. To further aggravate the deregulation in the pathogen, we have additionally deleted the GPR1 gene, encoding the nutrient receptor that activates the cyclic AMP-protein kinase A signaling pathway, which negatively regulates trehalose accumulation in yeasts. A gpr1 mutant is strongly affected in morphogenesis on solid media as well as in vivo in a mouse model but has only a slightly decreased virulence. The gpr1 tps2 double mutant, on the other hand, is completely avirulent in a mouse model for systemic infection. This strain accumulates very high T6P levels under stress conditions and has a growth defect at higher temperatures. We also show that a tps2 mutant is more sensitive to being killed by macrophages than the wild type or the gpr1 mutant. A double mutant has susceptibility similar to that of the single tps2 mutant. For morphogenesis on solid media, on the other hand, the gpr1 tps2 mutant shows a phenotype similar to that of the single gpr1 mutant. Taken together these results show that there is synergism between Gpr1 and Tps2 and that their combined inactivation results in complete avirulence. Combination therapy targeting both proteins may prove highly effective against pathogenic fungi with increased resistance to the currently used antifungal drugs.

The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans.

We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein-coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the Galpha protein Gpa2 or of the catalytic protein kinase A subunit TPK1 restores the wild-type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the mitogen-activated protein kinase pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-protein kinase A (PKA) pathway. We also show that, in the presence of glucose, CaGpr1 is important for amino acid-induced transition from yeast to hyphal cells. Finally, as opposed to previous reports, we show that CaGpa2 acts downstream of CaGpr1 as activator of the cAMP-PKA pathway but that deletion of neither CaGpr1 nor CaGpa2 affects glucose-induced cAMP signaling. In contrast, the latter is abolished in strains lacking CaCdc25 or CaRas1, suggesting that the CaCdc25-CaRas1 rather than the CaGpr1-CaGpa2 module mediates glucose-induced cAMP signaling in C. albicans.