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.     

Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism

Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast-like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site-directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.     

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.     

An IQGAP-related protein,encoded by AgCYK1, is required for septation in the filamentous fungus Ashbya gossypii

In filamentous ascomycetes hyphae are compartmentalized by septation in which the cytoplasm of the compartments are interconnected via septal pores. Thus, septation in filamentous fungi is different from cytokinesis in yeast like fungi. We have identified an Ashbya gossypii orthologue of the Saccharomyces cerevisiae CYK1 gene which belongs to the IQGAP-protein family. In contrast to S. cerevisiae disruption of AgCYK1 yields viable mutant strains that exhibit wildtype-like polarized hyphal growth rates. In the Agcyk1 mutant cortical actin patches localize to growing hyphal tips like wildtype, however, mutant hyphae are totally devoid of actin rings at presumptive septal sites. Septation in wildtype results in the formation of chitin rings. Agcyk1 mutant hyphae are aseptate and do not accumulate chitin in their cell walls. Agcyk1 mutant strains are completely asporogenous indicating that septation is essential for the formation of sporangia in A. gossypii. AgCyk1p-GFP localizes to sites of future septation as a ring prior to chitin depositioning. Furthermore, decrease in Cyk1p-ring diameter was found to be a prerequisite for the accumulation of chitin and septum formation.     

Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.     

Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog.

Pseudohyphal differentiation, a filamentous growth form of the budding yeast Saccharomyces cerevisiae, is induced by nitrogen starvation. The mechanisms by which nitrogen limitation regulates this process are currently unknown. We have found that GPA2, one of the two heterotrimeric G protein alpha subunit homologs in yeast, regulates pseudohyphal differentiation. Deltagpa2/Deltagpa2 mutant strains have a defect in pseudohyphal growth. In contrast, a constitutively active allele of GPA2 stimulates filamentation, even on nitrogen-rich media. Moreover, a dominant negative GPA2 allele inhibits filamentation of wild-type strains. Several findings, including epistasis analysis and reporter gene studies, indicate that GPA2 does not regulate the MAP kinase cascade known to regulate filamentous growth. Previous studies have implicated GPA2 in the control of intracellular cAMP levels; we find that expression of the dominant RAS2(Gly19Val) mutant or exogenous cAMP suppresses the Deltagpa2 pseudohyphal defect. cAMP also stimulates filamentation in strains lacking the cAMP phosphodiesterase PDE2, even in the absence of nitrogen starvation. Our findings suggest that GPA2 is an element of the nitrogen sensing machinery that regulates pseudohyphal differentiation by modulating cAMP levels.     

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white.

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.     

The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear. We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation. In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth. Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations. The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway. Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function. In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects. We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.     

Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS.

Diploid S. cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen. Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium. Pseudohyphal growth allows yeast cells to forage for nutrients. Pseudohyphal growth requires the polar budding pattern of a/alpha diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition. Constitutive activation of RAS2 or mutation of SHR3, a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSR1/BUD1 that causes random budding suppresses pseudohyphal growth.     

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.     

A novel endoplasmic reticulum membrane protein Rcr1 regulates chitin deposition in the cell wall of Saccharomyces cerevisiae

Congo red binds to the cell wall and inhibits the growth of yeast. In a screening for multicopy suppressor genes of Congo red hypersensitivity of erd1Delta mutant, we found that a previously uncharacterized gene, YBR005w, makes most of the Saccharomyces cerevisiae strains resistant to Congo red. This gene was named RCR1 (resistance to Congo red 1). An rcr1Delta null mutant showed an increased sensitivity to Congo red. RCR1 encodes a novel ER membrane protein with a single transmembrane domain. Molecular dissection suggested that the transmembrane domain and a part of the C-terminal polypeptide are sufficient for the activity. We examined the effect of RCR1 in various null mutants of genes related to the cell wall. The resistance of mutants to Congo red correlates with a reduction of chitin content. Multicopy RCR1 caused a significant decrease in the chitin content while the amount of alkali-soluble glucan did not change. The binding of Calcofluor white to the cell wall significantly decreased in these cells. Our results show that RCR1 regulates the chitin deposition and add firm genetic and biochemical evidences that the primary target of Congo red is chitin in S. cerevisiae.     

Mutational Analysis of Morphologic Differentiation in Saccharomyces Cerevisiae

A genetic analysis was undertaken to investigate the mechanisms controlling cellular morphogenesis in Saccharomyces cerevisiae. Sixty mutant strains exhibiting abnormally elongated cell morphology were isolated. The cell elongation phenotype in at least 26 of the strains resulted from a single recessive mutation. These mutations, designated generically elm (elongated morphology), defined 14 genes; two of these corresponded to the previously described genes GRR1 and CDC12. Genetic interactions between mutant alleles suggest that several ELM genes play roles in the same physiological process. The cell and colony morphology and growth properties of many elm mutant strains are similar to those of wild-type yeast strains after differentiation in response to nitrogen limitation into the pseudohyphal form. Each elm mutation resulted in multiple characteristics of pseudohyphal cells, including elongated cell shape, delay in cell separation, simultaneous budding of mother and daughter cells, a unipolar budding pattern, and/or the ability to grow invasively beneath the agar surface. Mutations in 11 of the 14 ELM gene loci potentiated pseudohyphal differentiation in nitrogen-limited medium. Thus, a subset of the ELM genes are likely to affect control or execution of a defined morphologic differentiation pathway in S. cerevisiae.     

KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties

The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall beta-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of beta-1,6-glucan and more chitin and beta-1,3-glucan and less mannoprotein than the WT. The remaining beta-1,6-glucan, about 20% of WT levels, exhibits a beta-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a beta-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall beta-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.     

真菌几丁质合酶的研究进展

真菌细胞壁几丁质的合成是一个复杂的过程, 其关键酶为几丁质合酶(CS)。近年来, 丝状真菌中的CS研究有了大的突破, 与酿酒酵母中只有3种CS不同, 丝状真菌中存在7种类别的CS。大部分临床和农业中重要的病原真菌都是丝状真菌, 文中对真菌中7种类别CS的结构和功能作了概述, 重点讨论了丝状真菌中重要的CS类别, 并介绍了CS作为抗真菌药物有效靶标的研究现状, 旨在为研究真菌CS及其抑制剂提供参考。