Deletion of the Candida albicans G-protein-coupled receptor, encoded by orf19.1944 and its allele orf19.9499, produces mutants defective in filamentous growth

Filamentous growth of Candida albicans occurs in response to a variety of environmental signals. The C. albicans gene orf19.1944 and its allele orf19.9499 are identical and are predicted to encode an 823-residue, 7-transmembrane-domain protein that has all the expected features of a G-protein-coupled receptor. The protein is 20.9% identical to the Saccharomyces cerevisiae Gpr1p receptor that signals both glucose availability and nitrogen limitation. Deletion of both copies of the gene in C. albicans abolished filamentation by colonies embedded in rich media (YPS, YPGal, and YPGlu), whereas mutants carrying a single copy of the gene were indistinguishable from the parental strain under these conditions. On medium containing low concentrations of ammonia (SLAD and SLAM media), surface colonies of both the homozygous deletion mutants and the mutants carrying a single copy of the gene were defective in filamentation. Serum-induced germ tube formation was unaffected by deletion of this gene, as was filamentation of the mutants growing on the surface of solid Spider medium at 37 degrees C or embedded in solid Spider medium at 25 degrees C. The protein encoded by orf19.1944 and orf19.9499 has a role in filamentation by both surface and embedded colonies, presumably as a sensor of environmental cues.     

Investigating the function of Ddr48p in Candida albicans

Candidiasis now represents the fourth most frequent nosocomial infection both in the United States and worldwide. Candida albicans is an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad-spectrum antibiotics, and other immune defects. The pathogenic potential of C. albicans is intimately related to certain key processes, including biofilm formation and filamentation. Ddr48p is a damage response protein that is significantly upregulated during both biofilm formation and filamentation, but its actual function is unknown. Previous studies have indicated that this protein may be essential in C. albicans but not Saccharomyces cerevisiae. Here we examined the function of Ddr48p and investigated the role of this protein in biofilm formation and filamentation. We demonstrated that this protein is not essential in C. albicans and appears to be dispensable for filamentation. However, DDR48 is required for the flocculation response stimulated by 3-aminotriazole-induced amino acid starvation. Furthermore, we examined the response of this deletion strain to a wide variety of environmental stressors and antifungal compounds. We observed several mild sensitivity or resistance phenotypes and also found that Ddr48p contributes to the DNA damage response of C. albicans. The results of this study reveal that the role of this highly expressed protein goes beyond a general stress response and impinges on a key facet of pathogenesis, namely, the ability to sense and respond to changes in the host environment.     

Involvement of Candida albicans pyruvate dehydrogenase complex protein X (Pdx1) in filamentation

For 50 years, physiologic studies in Candida albicans have associated fermentation with filamentation and respiration with yeast morphology. Analysis of the mitochondrial proteome of a C. albicans NDH51 mutant, known to be defective in filamentation, identified increased expression of several proteins in the respiratory pathway. Most notable was a 15-fold increase in pyruvate dehydrogenase complex protein X (Pdx1), an essential component of the pyruvate dehydrogenase complex. In basal salts medium with < or = 100 mM glucose as carbon source, two independent pdx1 mutants displayed a filamentation defect identical to ndh51; reintegration of one PDX1 allele restored filamentation. Concentrations of glucose < or = 100 mM did not correct the filamentation defect. Expanding on previous work, these studies suggest that increased expression of proteins extraneous to the electron transport chain compensates for defects in the respiratory pathway to maintain yeast morphology. Mitochondrial proteomics can aid in the identification of C. albicans genes not previously implicated in filamentation.     

Invasive filamentous growth of Candida albicans is promoted by Czf1p-dependent relief of Efg1p-mediated repression

Filamentation of Candida albicans occurs in response to many environmental cues. During growth within matrix, Efg1p represses filamentation and Czf1p relieves this repression. We propose that Czf1p interacts with Efg1p, altering its function. The complex regulation of filamentation may reflect the versatility of C. albicans as a pathogen.     

RIM101-dependent and-independent pathways govern pH responses in Candida albicans

Growth and differentiation of Candida albicans over a broad pH range underlie its ability to infect an array of tissues in susceptible hosts. We identified C. albicans RIM101, RIM20, and RIM8 based on their homology to components of the one known fungal pH response pathway. PCR product-disruption mutations in each gene cause defects in three responses to alkaline pH: filamentation, induction of PRA1 and PHR1, and repression of PHR2. We find that RIM101 itself is an alkaline-induced gene that also depends on Rim20p and Rim8p for induction. Two observations indicate that a novel pH response pathway also exists. First, PHR2 becomes an alkaline-induced gene in the absence of Rim101p, Rim20p, or Rim8p. Second, we created strains in which Rim101p activity is independent of Rim20p and Rim8p; in these strains, filamentation remains pH dependent. Thus, pH governs gene expression and cellular differentiation in C. albicans through both RIM101-dependent and RIM101-independent pathways.     

A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins.     

Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae.

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control.     

Effects of deletion mutations in the yeast Ces1 protein on cell growth and morphology and on high copy suppression of mutations in mRNA capping enzyme and translation initiation factor 4A.

The homologous Saccharomyces cerevisiae genes CES1 and CES4 act as high copy suppressors of temperature-sensitive mutations of Ceg1p, the yeast mRNA capping enzyme. Neither CES1 nor CES4 is essential for cell growth. We find that a double deletion mutant (Delta ces1 Delta ces4 ) grows at 25-37 degrees C, but not at 16 degrees C. Delta ces1 Delta ces4 cells display gross defects in cell shape and budding even at permissive temperatures. Functional analysis of CES1 deletion mutants defines a 145 amino acid C-terminal segment of the 915 amino acid Ces1 protein that is necessary and sufficient to complement the Delta ces1 Delta ces 4 cold-sensitive phenotype, to restore normal morphology and to suppress the temparature-sensitive mutant ceg1-25 . A 147 amino acid C-terminal segment of the 942 amino acid Ces4 protein is sufficient to carry out these same functions. Within this carboxyl domain Ces1p and Ces4p are 80% identical to one another. We report isolation of CES1 in a separate screen for high copy suppression of a temperature-sensitive mutation (A79V) of the yeast translation initiation factor Tif1p (eIF-4A). Deletion of the N-terminal 249 amino acids of Ces1p abolished tif1-A79V suppressor function. CES4 on a multicopy plasmid was unable to suppress tif1-A79V . We surmise that whereas the carboxyl domains of Ces1p and Ces4p are functionally redundant in controlling cell morphology and in suppressing ceg1-25 , full-length Ces1p and Ces4p evince distinct genetic interactions that are likely mediated by their N-terminal segments.     

Identification of an exoribonuclease homolog, CaKEM1/CaXRN1, in Candida albicans and its characterization in filamentous growth

KEM1/XRN1 and RAT1 are two known exoribonuclease genes in Saccharomyces cereivsiae and encode a cytoplasmic and nuclear exoribonuclease, respectively. CaKEM1/CaXRN1 and CaRAT1, the Candida albicans homologs of 5'-->3' exoribonuclease genes, were identified by protein sequence comparisons and by functional complementation of the S. cerevisiae kem1/xrn1 null mutation. The deduced amino acid sequences of CaKEM1 and CaRAT1 show 51% and 55% identities to those of the S. cerevisiae KEM1 and RAT1, respectively. The exonuclease motifs were found to be highly conserved in CaKem1p and CaRat1p. We disrupted two chromosomal copies of CaKEM1 in a diploid C. albicans strain and demonstrate that C. albicans kem1/kem1 mutants are defective in filamentous growth on filamentous-inducing media. These results imply that CaKEM1 is involved in filamentous growth of C. albicans.     

Protein arginine methylation in Candida albicans: role in nuclear transport

Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and omega-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3Delta strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2Delta/rmt2Delta strain, as well as biochemical evidence for additional cryptic PRMTs.