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.     

A G1 Cyclin Is Necessary for Maintenance of Filamentous Growth in Candida albicans

Candida albicans undergoes a dramatic morphological transition in response to various growth conditions. This ability to switch from a yeast form to a hyphal form is required for its pathogenicity. The intractability of Candida to traditional genetic approaches has hampered the study of the molecular mechanism governing this developmental switch. Our approach is to use the more genetically tractable yeast Saccharomyces cerevisiae to yield clues about the molecular control of filamentation for further studies in Candida. G1 cyclins Cln1 and Cln2 have been implicated in the control of morphogenesis in S. cerevisiae. We show that C. albicans CLN1 (CaCLN1) has the same cell cycle-specific expression pattern as CLN1 and CLN2 of S. cerevisiae. To investigate whether G1 cyclins are similarly involved in the regulation of cell morphogenesis during the yeast-to-hypha transition of C. albicans, we mutated CaCLN1. Cacln1/Cacln1 cells were found to be slower than wild-type cells in cell cycle progression. The Cacln1/Cacln1 mutants were also defective in hyphal colony formation on several solid media. Furthermore, while mutant strains developed germ tubes under several hypha-inducing conditions, they were unable to maintain the hyphal growth mode in a synthetic hypha-inducing liquid medium and were deficient in the expression of hypha-specific genes in this medium. Our results suggest that CaCln1 may coordinately regulate hyphal development with signal transduction pathways in response to various environmental cues.     

Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace (fgf8) and coincident defects in CNS development

During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline. The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.     

Effects of anoxia and the mitochondrion on expression of aerobic nuclear COX genes in yeast: evidence for a signaling pathway from the mitochondrial genome to the nucleus

Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes. Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated. Recently, it has been reported that induction of some hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process. In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeast aerobic nuclear COX genes. We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclear cox mutants and mitochondrial rho(0) and mit(-) mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions. This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase. We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another. These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclear COX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.     

Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes

The yeast Candida albicans is the most important fungal pathogen of humans and a model organism for studying fungal virulence. Sequencing of the C. albicans genome will soon be completed, allowing systematic approaches to analyse gene function. However, techniques to define and characterize essential genes in this permanently diploid yeast are limited. We have developed an efficient method to create conditional lethal C. albicans null mutants by inducible, FLP-mediated gene deletion. Both wild-type alleles of the CDC42 or the BEM1 gene were deleted in strains that carried an additional copy of the respective gene that could be excised from the genome by the site-specific recombinase FLP. Expression of a C. albicans-adapted FLP gene under the control of an inducible promoter generated cell populations consisting of > or = 99.9% null mutants. Upon plating, these cells were unable to form colonies, demonstrating that CDC42 and BEM1 are essential genes in C. albicans. The cdc42 null mutants failed to produce buds and hyphae and grew as large, round cells instead, suggesting that they lacked the ability to produce polarized cell growth. However, the cells still responded to hyphal inducing signals by aggregating and expressing hypha-specific genes, behaviours typical of the mycelial growth form of C. albicans. Budding cells and germ tubes of bem1 null mutants exhibited morphological abnormalities, demonstrating that BEM1 is essential for normal growth of both yeast and hyphae. Inducible, FLP-mediated gene deletion provides a powerful approach to generate conditional lethal C. albicans mutants and allows the functional analysis of essential genes.