Combined Inactivation of the Candida albicans GPR1 and TPS2 Genes Results in Avirulence in a Mouse Model for Systemic Infection

Abstract

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.Trehalose is known as a stress-protective sugar synthesized under unfavorable growth conditions in a variety of microorganisms (4). It can be synthesized by several pathways (1), but in yeasts it is made only in a two-step process carried out by trehalose-6-phosphate (T6P) synthase (Tps1) and T6P phosphatase (Tps2). Tps1 catalyzes the conversion of UDP-glucose and glucose-6-phosphate into T6P. The second enzyme, Tps2, dephosphorylates T6P to produce trehalose. In Saccharomyces cerevisiae, these enzymes have been studied extensively (4). Apart from a catalytic function in the biosynthesis of trehalose, the ScTps1 enzyme also plays a crucial role in controlling the influx of glucose into glycolysis (5). As a consequence, a yeast tps1⌬ mutant is not able to grow on glucose because of a very fast deregulation of metabolism due to strong accumulation of sugar phosphates in the first part of glycolysis (31). Deletion of the yeast TPS2 gene results in a thermosensitivity phenotype, which is also caused by deregulation of trehalose metabolism. At higher temperatures (or under any other stress condition), the Sctps2⌬ mutant accumulates a high level of T6P, resulting in rapid deprivation of free phosphate (9, 28). Because the enzymes of trehalose metabolism are not present in humans and because trehalose is an important stress-protecting molecule in many fungi, these enzymes have been investigated as potential targets fo...

Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and Reduces In Vivo Pathogenicity

“…Mice were infected via the lateral caudal vein with 0.1 ml of a C. albicans suspension containing 1 ϫ 10 6 cells (17). Animal survival was evaluated daily (18).…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Biochemical studies revealed that Gpr1 and Gpa2 are required for the glucose-dependent increase in intracellular cAMP levels (386), and they are important for amino-acid-induced morphogenesis in the presence of glucose (358). Although gpr1 and gpa2 homozygous deletion mutants are fully virulent in the mouse (386), the combined deletion of GPR1 and TPS2, encoding the trehalose-6-phosphate phosphatase, results in avirulence in a mouse model of infection (356). Ras1 is a guanine nucleotide binding protein which acts downstream of Mep2.…”

Tight Control of Trehalose Content Is Required for Efficient Heat-induced Cell Elongation in Candida albicans

“…Active Growth in C. albicans-Previous findings have shown that absence of GPR1 in C. albicans leads to increased trehalose content as well as activity of the Tps1 enzyme, especially in heat shock conditions (30). We investigated the role of Gpr1 on trehalose content in C. albicans in conditions of 37°C, the physiological temperature that is not experienced as a stress in the pathogenic fungus.…”

“…As gpr1⌬/gpr1⌬ cells accumulate trehalose even at 30°C while actively growing (data not shown and Ref. 30), we investigated whether geldanamycin was able to induce the filamentation phenotype in the mutant strain. Whereas extrusions of the cells were readily observed in wild type upon addition of geldanamycin at the noninducing temperature of 30°C, the gpr1⌬/gpr1⌬ mutant was partially defective in this response (Fig.…”

“…Despite the high degree of sequence homology between the two, their functions have diverged substantially, the most striking difference being the apparent loss of its role in fast glucose signaling to adenylate cyclase for cAMP production (7). A mild induction of TPS activity at 43°C in the gpr1⌬/gpr1⌬ strain and avirulence of a gpr1⌬/gpr1⌬ tps2⌬/tps2⌬ strain suggested a connection between the G protein-coupled receptor and trehalose metabolism, a feature suggested to be downstream of PKA (30). It is now clear from the present findings that these phenotypes were caused by the role of Gpr1 in negatively regulating the trehalose biosynthesis activity when cells are actively growing under optimal conditions and do not require trehalose as a compatible solute.…”