We are studying the function of host extracellular matrix components in the pathogenesis of Candida albicans. Fibronectin mediates adhesion of C. albicans through two receptors expressed by the pathogen. A high affinity receptor recognizes the collagen-binding domain of fibronectin, and a low affinity receptor recognizes the cell-binding domain of fibronectin. Expression of both fibronectin receptors is tightly regulated by growth conditions. We previously identified hemoglobin as a highly specific inducer of the low affinity receptor. Hemoglobin-inducible binding was observed in all clinical isolates of C. albicans and in other pathogenic members of the Candida genus. The ability to respond to hemoglobin may play an important role in pathogenesis, since only pathogenic strains of C. albicans express hemolytic activity that can release hemoglobin from erythrocytes. Hemoglobin may therefore be a host environmental cue that triggers extracellular matrix receptor expression at a septic site. In addition to regulating expression of adhesion receptors on the yeast, we have identified several genes whose expression is induced by hemoglobin. Inhibitors of signaling pathways stimulated by hemoglobin may decrease the pathogenicity of C. albicans in immunocompromised cancer patients and thereby prevent disseminated candidemia.We identified a gene induced following growth in the presence of exogenous hemoglobin, HBR1, that functions as a repressor of white-opaque switching and mating. Suppressing switching during vascular dissemination of the organism may be advantageous because opaque cells are more susceptible to host defenses. However, deletion of both alleles of this gene give a lethal phenotype, suggesting that Hbr1 has additional functions in C. albicans and may be a useful target for a new class of antifungal therapeutics. Hbr1p contains a predicted phosphate binding loop (P-loop), a highly conserved motif found in ATP- and GTP- binding proteins. We investigated nucleotide binding to Hbr1p and found specific binding of ATP with Kd = 2 iM. ATP binding induced a conformational change that protected Hbr1p from proteolysis by trypsin, whereas other nucleotides offered no protection. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and less conserved glycine (G19S) decreased binding affinity for ATP. ATP bound more avidly than ATP?S are observed for wild-type and mutant Hbr1p. Hbr1p contains some sequences characteristic of adenylate kinases, but assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative.Since HBR1 heterozygous cells can function as de facto MTL 'a' mating strains, we examined opaque and white cells of the HBR1 +/- strain CAMP 43 for regulation of genes involved in the mating response using quantitative RT-PCR. We uncovered a series of genes regulated in a manner other than that characterized in a/a cells generated by MTLa1a2 deletions. Components of the mating pheromone pathway defined in a/a cells as opaque-specific, pheromone responsive STE2 (a-factor receptor) and STE4 (G protein a subunit) were 300- and 1300-fold increased in CAMP43 opaque cells in absence of a mating pheromone. Several genes defined as switching dependent and pheromone independent, including ICL1 and YEL007, were regulated identical to those defined in a/a strains. On the other hand, the expression of two genes necessary for 'a' mating pheromone maturation and secretion, RAM2 and HST6, was decreased 32- and 8-fold, respectively, in opaque CAMP 43 cells, indicating that the ability to secrete 'a' mating pheromone is under positive control by Hbr1p. Hyphal-specific genes responsive to a pheromone and involved in mating projection formation RBT1, RBT4, ECE1, SAP5, HWP1, and CPH1 remained unchanged, suggesting that these are Hbr1p independent.
