Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

Analysis of phase-specific gene expression at the single-cell level in the white-opaque switching system of Candida albicans

The opportunistic fungal pathogen Candida albicans can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and evasion of host defense mechanisms. Phenotypic switching has been studied intensively for the white-opaque switching system of strain WO-1. To facilitate the molecular analysis of phenotypic switching, we have constructed homozygous ura3 mutants from strain WO-1 by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid (MPA) resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. To investigate a possible cell type-independent switching in the expression of individual phase-specific genes, two different reporter genes that allowed the analysis of gene expression at the single-cell level were integrated into the genome, using URA3 as a selection marker. Fluorescence microscopic analysis of cells in which a GFP reporter gene was placed under the control of phase-specific promoters demonstrated that the opaque-phase-specific SAP1 gene was detectably expressed only in opaque cells and that the white-phase-specific WH11 gene was detectably expressed only in white cells. When MPA(R) was used as a reporter gene, it conferred an MPA-resistant phenotype on opaque but not white cells in strains expressing it from the SAP1 promoter, which was monitored at the level of single cells by a significantly enlarged size of the corresponding colonies on MPA-containing indicator plates. Similarly, white but not opaque cells became MPA resistant when MPA(R) was placed under the control of the WH11 promoter. The analysis of these reporter strains showed that cell type-independent phase variation in the expression of the SAP1 and WH11 genes did not occur at a detectable frequency. The expression of these phase-specific genes of C. albicans in vitro, therefore, is tightly linked to the cell type.     

Alpha-pheromone-induced "shmooing" and gene regulation require white-opaque switching during Candida albicans mating

A 14-mer α-pheromone peptide of Candida albicans was chemically synthesized and used to analyze the role of white-opaque switching in the mating process. The α-pheromone peptide blocked cell multiplication and induced “shmooing” in a/a cells expressing the opaque-phase phenotype but not in a/a cells expressing the white-phase phenotype. The α-pheromone peptide induced these effects at 25°C but not at 37°C. An analysis of mating-associated gene expression revealed several categories of gene regulation, including (i) MTL-homozygous-specific, pheromone stimulated, switching-independent (CAG1 and STE4); (ii) mating type-specific, pheromone-induced, switching-independent (STE2); and (iii) pheromone-induced, switching-dependent (FIG1, KAR4, and HWP1). An analysis of switching-regulated genes revealed an additional category of opaque-phase-specific genes that are downregulated by α-pheromone only in a/a cells (OP4, SAP1, and SAP3). These results demonstrate that α-pheromone causes shmooing, the initial step in the mating process, only in a/a cells expressing the opaque phenotype and only at temperatures below that in the human host. These results further demonstrate that although some mating-associated genes are stimulated by the α-pheromone peptide in both white- and opaque-phase cells, others are stimulated only in opaque-phase cells, revealing a category of gene regulation unique to C. albicans in which α-pheromone induction requires the white-opaque transition. These results demonstrate that in C. albicans, the mating process and associated gene regulation must be examined within the context of white-opaque switching.     

Regulatory Diversity of TUP1 in Cryptococcus neoformans

Cryptococcus neoformans serotype A strains, the major cause of cryptococcosis, are distributed worldwide, while serotype D strains are more concentrated in Central Europe. We have previously shown that deletion of the global regulator TUP1 in serotype D isolates results in a novel peptide-mediated, density-dependent growth phenotype that mimics quorum sensing and is not known to exist in other fungi. Unlike for tup1Δ strains of serotype D, the density-dependent growth phenotype was found to be absent in tup1Δ strains of serotype A which had been derived from several different genetic clusters. The serotype A H99 tup1Δ strain showed less retardation in the growth rate than tup1Δ strains of serotype D, but the mating efficiency was found to be similar in both serotypes. Deletion of TUP1 in the H99 strain resulted in significantly enhanced capsule production and defective melanin formation and also revealed a unique regulatory role of the TUP1 gene in maintaining iron/copper homeostasis. Differential expression of various genes involved in capsule formation and iron/copper homeostasis was observed between the wild-type and tup1Δ H99 strains. Furthermore, the H99 tup1Δ strain displayed pleiotropic effects which included sensitivity to sodium dodecyl sulfate, susceptibility to fluconazole, and attenuated virulence. These results demonstrate that the global regulator TUP1 has pathobiological significance and plays both conserved and distinct roles in serotype A and D strains of C. neoformans.The fungal Tup1 proteins function as global repressors which regulate a large number of genes associated with growth, morphological differentiation, and sexual and asexual reproduction. As a consequence, tup1 mutants are known to display numerous phenotypes (9, 19, 42). The deletion of TUP1 in Candida albicans results in constitutive filamentous growth with no budding yeast cells and is accompanied by loss of virulence (2, 32). In Penicillium marneffei, the only dimorphic species known in the genus Penicillium, deletion of the TUP1 homolog, tupA, confers reduced filamentation and abnormality in yeast morphogenesis (38). In the filamentous fungi Aspergillus nidulans and Neurospora crassa, deletion of the TUP1 homologs, rcoA and rco-1, respectively, severely affects growth and sexual and asexual reproduction (12, 46).Cryptococcus neoformans is a bipolar heterothallic basidiomycetous yeast with two serotypes, A and D, and the function of Tup1 has been studied only for serotype D strains (26, 27). While disruption of TUP1 in strains of serotype D did not affect yeast or hyphal cell morphology, it resulted in mating-type-dependent differences, including temperature-dependent growth, sensitivity to 0.8 M KCl, and expression of genes in several other biological pathways (26). Most importantly, tup1Δ strains displayed a peptide-mediated quorum-sensing-like phenomenon in both mating types of serotype D strains which has not been reported for any other fungal species (27).According to genome sequence data, the serotype A reference strain H99 shares 95% sequence identity with the serotype D reference strain JEC21 (29). However, serotype-specific differences between the two strains have been demonstrated in two major signaling pathways, the pheromone-responsive Cpk1 mitogen-activated protein kinase and cyclic AMP (cAMP) (5, 13, 41, 47). In addition, the high-osmolarity glycerol (HOG) pathway also showed regulatory disparity between the two serotypes (1, 8). Since the regulation of peptide-mediated quorum sensing by TUP1 is reported only for serotype D strains, we sought to determine whether the deletion of TUP1 in serotype A strains would have similar consequences. Surprisingly, we found striking differences in the phenotypes manifested by tup1Δ strains of the two serotypes. We report here the serotype-specific differences in TUP1 regulation between A and D strains and the novel regulatory role of TUP1 in maintaining iron/copper homeostasis in C. neoformans.     

Antagonistic interplay of Swi1 and Tup1 on filamentous growth of Candida albicans

Candida albicans is a polymorphic human opportunistic pathogen in which the Swi-Snf complex functions as an activator whereas Tup1 acts as a general repressor during the yeast-hyphae transition. In Saccharomyces cerevisiae, the interplay between the Swi-Snf complex and the Tup1-Ssn6 repressive complex regulates the balance between active and repressed chromatin structures of a number of genes. To study the interplay between Candida albicans Swi1 and Tup1 and their effects on morphogenesis, we analyzed phenotypes of swi1/swi1, tup1/tup1 and swi1/swi1 tup1/tup1 mutants under various growth conditions. The swi1/swi1 mutant failed to form true hyphae, whereas the tup1/tup1 mutant exhibited constitutive filamentous growth. Deletion of SWI1 in the tup1/tup1 mutant completely blocked hyphal growth under all the conditions examined. Under aerobic conditions, the swi1/swi1 tup1/tup1 mutant most resembled the swi1/swi1 mutant in phenotype, actin polarization and gene expression pattern. In invaded agar, the double mutant showed similar phenotypes as the swi1/swi1 mutant, while under embedded conditions, it grew as a pseudohypha-like form different from that of the wild-type strain, swi1/swi1 or tup1/tup1 mutants. These results suggest that Swi1 may play a dominant role by antagonizing the repressive effect of the Tup1 on hyphal development in C. albicans.     

tailup, a LIM-HD gene, and Iro-C cooperate in Drosophila dorsal mesothorax specification

The LIM-HD gene tailup (tup; also known as islet) has been categorised as a prepattern gene that antagonises the formation of sensory bristles on the notum of Drosophila by downregulating the expression of the proneural achaete-scute genes. Here we show that tup has an earlier function in the development of the imaginal wing disc; namely, the specification of the notum territory. Absence of tup function causes cells of this anlage to upregulate different wing-hinge genes and to lose expression of some notum genes. Consistently, these cells differentiate hinge structures or modified notum cuticle. The LIM-HD co-factors Chip and Ssdp are also necessary for notum specification. This suggests that Tup acts in this process in a complex with Chip and Ssdp. Overexpression of tup, together with araucan, a 'pronotum' gene of the iroquois complex (Iro-C), synergistically reinforces the weak capacity of either gene, when overexpressed singly, to induce ectopic notum-like development. Whereas the Iro-C genes are activated in the notum anlage by EGFR signalling, tup is positively regulated by Dpp signalling. Our data support a model in which the EGFR and Dpp signalling pathways, with their respective downstream Iro-C and tup genes, converge and cooperate to commit cells to the notum developmental fate.