Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p

The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Delta mutants for growth at 37 degrees C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans.     

The GRR1 gene of Candida albicans is involved in the negative control of pseudohyphal morphogenesis

The opportunistic fungal pathogen Candida albicans can grow as yeast, pseudohyphae or true hyphae. C. albicans can switch between these morphologies in response to various environmental stimuli and this ability to switch is thought to be an important virulence trait. In Saccharomyces cerevisiae, the Grr1 protein is the substrate recognition component of an SCF ubiquitin ligase that regulates cell cycle progression, cell polarity and nutrient signaling. In this study, we have characterized the GRR1 gene of C. albicans. Deletion of GRR1 from the C. albicans genome results in a highly filamentous, pseudohyphal morphology under conditions that normally promote the yeast form of growth. Under hypha-inducing conditions, most cells lacking GRR1 retain a pseudohyphal morphology, but some cells appear to switch to hyphal-like growth and express the hypha-specific genes HWP1 and ECE1. The C. albicans GRR1 gene also complements the elongated cell morphology phenotype of an S. cerevisiae grr1Delta mutant, indicating that C. albicans GRR1 encodes a true orthologue of S. cerevisaie Grr1. These results support the hypothesis that the Grr1 protein of C. albicans, presumably as the F-box subunit of an SCF ubiquitin ligase, has an essential role in preventing the switch from the yeast cell morphology to a pseudohyphal morphology.     

The DNA binding protein Rfg1 is a repressor of filamentation in Candida albicans

We have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.     

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.     

白念珠菌CaPPE1的克隆及其在酿酒酵母形态发生中的功能研究

白念珠茵的致病性与其形态转变相关,白念珠茵的形态转换受各种外界信号和细胞内信号转导途径的调控。转录因子Flo8在酿酒酵母形态发生中起重要作用,我们将白念珠茵基因组文库导入flo8缺失株中,筛选能够校正flo8缺失株侵入生长缺陷的基因,分离得到一个与酿酒酵母蛋白磷酸酯酶甲基酯酶PPEI同源的基因,命名为CaPPEl。CaPPEl的基因编码区全长1083bp,推测编码一个361氨基酸的蛋白。在单倍体酿酒酵母中,CaPPE1基因的表达可以部分回复flo8缺失株的侵入生长缺陷,但是在MAPK途径缺失株中不能进行侵入生长。在双倍体酿酒酵母中,CaPPEl基因的表达可以部分激活MAPK途径成员缺失株的茵丝生长缺陷,但却只能在flo8缺失株中产生微弱的激活作用。结果表明CaPpel在酿酒酵母的假茵丝生长和侵入生长中参与的信号转导途径不同。     

Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells.

The ability to switch between yeast and hyphal morphologies is an important virulence factor for the opportunistic pathogen Candida albicans. Although the kinetics of appearance of the filamentous ring that forms at the incipient septum differ in yeast and cells forming hyphae (germ tubes) (), the molecular mechanisms that regulate this difference are not known. Int1p, a C. albicans gene product with similarity in its C terminus to Saccharomyces cerevisiae Bud4p, has a role in hyphal morphogenesis. Here we report that in S. cerevisiae, Int1p expression results in the growth of highly polarized cells with delocalized chitin and defects in cytokinesis and bud-site selection patterns, phenotypes that are also seen in S. cerevisiae septin mutant strains. Expression of high levels of Int1p in S. cerevisiae generated elaborate spiral-like structures at the periphery of the polarized cells that contained septins and Int1p. In addition, Int1p coimmunoprecipitated with the Cdc11p and Cdc12p septins, and Cdc12p is required for the establishment and maintenance of these Int1p/septin spirals. Although Swe1p kinase contributes to INT1-induced filamentous growth in S. cerevisiae, it is not required for the formation of ectopic Int1p/septin structures. In C. albicans, Int1p was important for the axial budding pattern and colocalized with Cdc3p septin in a ring at the mother-bud neck of yeast and pseudohyphal cells. Under conditions that induce hyphae, both Cdc3p and Int1p localized to a ring distal to the junction of the mother cell and germ tube. Thus, placement of the Int1p/septin ring with respect to the mother-daughter cell junction distinguishes yeast/pseudohyphal growth from hyphal growth in C. albicans.