Showing papers in "Eukaryotic Cell in 2003"

Engineered Control of Cell Morphology In Vivo Reveals Distinct Roles for Yeast and Filamentous Forms of Candida albicans during Infection

Abstract: It is widely assumed that the ability of Candida albicans to switch between different morphologies is required for pathogenesis. However, most virulence studies have used mutants that are permanently locked into either the yeast or filamentous forms which are avirulent but unsuitable for discerning the role of morphogenetic conversions at the various stages of the infectious process. We have constructed a strain in which this developmental transition can be externally modulated both in vitro and in vivo. This was achieved by placing one copy of the NRG1 gene (a negative regulator of filamentation) under the control of a tetracycline-regulatable promoter. This modified strain was then tested in an animal model of hematogenously disseminated candidiasis. Mice injected with this strain under conditions permitting hyphal development succumbed to the infection, whereas all of the animals injected under conditions that inhibited this transition survived. Importantly, fungal burdens were almost identical in both sets of animals, indicating that, whereas filament formation appears to be required for the mortality resulting from a deep-seated infection, yeast cells play an important role early in the infectious process by extravasating and disseminating to the target organs. Moreover, these infecting Candida yeast cells still retained their pathogenic potential, as demonstrated by allowing this developmental transition to occur at various time points postinfection. We demonstrate here the importance of morphogenetic conversions in C. albicans pathogenesis. This engineered strain should provide a useful tool in unraveling the individual contributions of the yeast and filamentous forms at various stages of the infectious process.

Molecular genetics of pathogenic oomycetes.

Abstract: Parasitic and pathogenic lifestyles have evolved repeatedly in eukaryotes ([93][1]). Several parasitic eukaryotes represent deep phylogenetic lineages, suggesting that they feature unique molecular processes for infecting their hosts. One such group is formed by the oomycetes. Traditionally, due to

trans and cis Splicing in Trypanosomatids: Mechanism, Factors, and Regulation

Abstract: mRNA maturation in trypanosomes differs from the process in most eukaryotes mainly because protein-coding genes are transcribed into polycistronic RNAs in this organism ([78][1]). Studies from the ongoing genome project suggest that the entire chromosome may be transcribed as large transcripts, but

The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans.

Abstract: Candida albicans mutants with mutations in mitogen-activated protein (MAP) kinase HOG1 displayed an increased sensitivity to agents producing reactive oxygen species, such as oxidants (menadione, hydrogen peroxide, or potassium superoxide), and UV light. Consistent with this finding, C. albicans Hog1 was activated not only in response to an increase in external osmolarity, as happens with its Saccharomyces cerevisiae homologue, but also in response to hydrogen peroxide. The Hog1-mediated response to oxidative stress was different from that of transcription factor Cap1, the homologue of S. cerevisiae Yap1, as shown by the different sensitivities to oxidants and the kinetics of cell death of cap1, hog1, and hog1 cap1 mutants. Deletion of CAP1 did not influence the level of Hog1 phosphorylation, and deletion of HOG1 did not affect Cap1 nuclear localization. Moreover, we show that the HOG1 gene plays a role in chlamydospore formation, another oxygenrelated morphogenetic event, as demonstrated by the fact that hog1 cells were unable to generate these thickwalled structures in several media through a mechanism different from that of the EFG1 regulator. This is the first demonstration of the role of the Hog1-mediated MAP kinase pathway in resistance to oxidative stress in pathogenic fungi, and it allows us to propose a molecular model for the oxidative stress response in C. albicans.

Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada

Abstract: Cryptococcus neoformans is a human fungal pathogen that exists as three distinct varieties or sibling species: the predominantly opportunistic pathogens C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A) and the primary pathogen C. neoformans var. gattii (serotypes B and C). While serotypes A and D are cosmopolitan, serotypes B and C are typically restricted to tropical regions. However, serotype B isolates of C. neoformans var. gattii have recently caused an outbreak on Vancouver Island in Canada, highlighting the threat of this fungus and its capacity to infect immunocompetent individuals. Here we report a large-scale analysis of the mating abilities of serotype B and C isolates from diverse sources and identify unusual strains that mate robustly and are suitable for further genetic analysis. Unlike most isolates, which are of both the a and α mating types but are predominantly sterile, the majority of the Vancouver outbreak strains are exclusively of the α mating type and the majority are fertile. In an effort to enhance mating of these isolates, we identified and disrupted the CRG1 gene encoding the GTPase-activating protein involved in attenuating pheromone response. crg1 mutations dramatically increased mating efficiency and enabled mating with otherwise sterile isolates. Our studies provide a genetic and molecular foundation for further studies of this primary pathogen and reveal that the Vancouver Island outbreak may be attributable to a recent recombination event.

Whole-Genome Analysis of Two-Component Signal Transduction Genes in Fungal Pathogens

Abstract: Two-component phosphorelay systems are minimally comprised of a histidine kinase (HK) component, which autophosphorylates in response to an environmental stimulus, and a response regulator (RR) component, which transmits the signal, resulting in an output such as activation of transcription, or of a mitogen-activated protein kinase cascade. The genomes of the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans encode one, three, and three HKs, respectively. In contrast, the genome sequences of the filamentous ascomycetes Neurospora crassa, Cochliobolus heterostrophus (Bipolaris maydis), Gibberella moniliformis (Fusarium verticillioides), and Botryotinia fuckeliana (Botrytis cinerea) encode an extensive family of two-component signaling proteins. The putative HKs fall into 11 classes. Most of these classes are represented in each filamentous ascomycete species examined. A few of these classes are significantly more prevalent in the fungal pathogens than in the saprobe N. crassa, suggesting that these groups contain paralogs required for virulence. Despite the larger numbers of HKs in filamentous ascomycetes than in yeasts, all of the ascomycetes contain virtually the same downstream histidine phosphotransfer proteins and RR proteins, suggesting extensive cross talk or redundancy among HKs.

Fatal Attraction: Nonself Recognition and Heterokaryon Incompatibility in Filamentous Fungi

Abstract: Vegetative incompatibility is a common phenomenon in filamentous fungi, including ascomycete, basidiomycete, and zygomycete fungi ([27][1], [70][2], [80][3]). A subset of vegetative incompatibility reactions includes events that require hyphal fusion and heterokaryon formation, whereby genetically

RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei.

Abstract: In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to α-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, αβ-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

The expression of sterigmatocystin and penicillin genes in Aspergillus nidulans is controlled by veA, a gene required for sexual development.

Abstract: Secondary metabolism is commonly associated with morphological development in microorganisms, including fungi. We found that veA, a gene previously shown to control the Aspergillus nidulans sexual/asexual developmental ratio in response to light, also controls secondary metabolism. Specifically, veA regulates the expression of genes implicated in the synthesis of the mycotoxin sterigmatocystin and the antibiotic penicillin. veA is necessary for the expression of the transcription factor aflR, which activates the gene cluster that leads to the production of sterigmatocystin. veA is also necessary for penicillin production. Our results indicated that although veA represses the transcription of the isopenicillin synthetase gene ipnA, it is necessary for the expression of acvA, the key gene in the first step of penicillin biosynthesis, encoding the delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase. With respect to the mechanism of veA in directing morphological development, veA has little effect on the expression of the known sexual transcription factors nsdD and steA. However, we found that veA regulates the expression of the asexual transcription factor brlA by modulating the α/β transcript ratio that controls conidiation.

A yeast under cover: the capsule of Cryptococcus neoformans.

Abstract: Few fungi are pathogenic to humans. Of these, Cryptococcus neoformans has emerged as an important cause of mortality in immunocompromised patients, especially those with AIDS. As a result, extensive research efforts have addressed the pathogenesis and virulence of this organism. C. neoformans is a basidiomycetous fungus that is ubiquitous in the environment, where it is found in soil, in association with certain trees, and in bird guano (16). Because of its ubiquity, it has been suggested that most people are exposed to C. neoformans early in life (41). The fungus is heterothallic, with mating types MATa and MAT. Asexual reproduction takes place either by budding or, in the case of MAT cells, by haploid fruiting in response to nutrient deprivation or exposure to the mating pheromone a factor (106). Sexual reproduction occurs when cells of opposite mating types come together to form a heterokaryon that ultimately leads to the production of basidia and basidiospores (64). Desiccated cells and the spores formed by haploid fruiting or sexual reproduction have all been suggested to serve as infective particles, which must be less than 2 m in diameter to penetrate the lung parenchyma (44, 86). Infection occurs when the fungal particles are inhaled and enter the alveolar space. In most immunocompetent individuals, this infection is either cleared or remains dormant until an immune imbalance leads to further development. In the setting of compromised immune function, however, the fungus disseminates, with particular tropism for the central nervous system. In severe cases, cryptococcal infection progresses to a meningoencephalitis that is fatal if left untreated. C. neoformans virulence is mediated predominantly by a polysaccharide capsule that surrounds its cell wall and has multiple effects on the host immune system. This structure provides a physical barrier that interferes with normal phagocytosis and clearance by the immune system. Capsule components inhibit the production of proinflammatory cytokines, deplete complement components (by efficiently binding them), and reduce leukocyte migration to sites of inflammation (11). The capsule also constitutes the major diagnostic feature of cryptococcosis, because its components can be detected in the bloodstream and it can be visualized with light microscopy by using India ink staining. The capsule excludes the ink particles and forms characteristic halos (Fig. 1A) whose diameters are often several times that of the cell. The elaborate structure of the capsule may also be appreciated by electron microscopy (Fig. 1B and C). Given the importance of the capsule in cryptococcal disease, tremendous effort has been applied in recent years to understanding its biology. This review focuses on the resulting advances in our understanding of the structure and synthesis of the capsular components, the incorporation of these components into the existing capsular network, the association between the capsule and the cell wall, and the regulation of capsule growth.

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei.

Abstract: Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

Mannitol Is Required for Stress Tolerance in Aspergillus niger Conidiospores

Abstract: d-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpdA gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpdA in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpdA null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a ΔmpdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions.

The yeast protein kinase C cell integrity pathway mediates tolerance to the antifungal drug caspofungin through activation of Slt2p mitogen-activated protein kinase signaling.

Abstract: The echinocandin caspofungin is a new antifungal drug that blocks cell wall synthesis through inhibition of beta-(1-3)-glucan synthesis. Saccharomyces cerevisiae cells are able to tolerate rather high caspofungin concentrations, displaying high viability at low caspofungin doses. To identify yeast genes implicated in caspofungin tolerance, we performed a genome-wide microarray analysis. Strikingly, caspofungin treatment rapidly induces a set of genes from the protein kinase C (PKC) cell integrity signaling pathway, as well as those required for cell wall maintenance and architecture. The mitogen-activated protein kinase Slt2p is rapidly activated by phosphorylation, triggering signaling through the PKC pathway. Cells lacking genes such as SLT2, BCK1, and PKC1, as well as the caspofungin target gene, FKS1, display pronounced hypersensitivity, demonstrating that the PKC pathway is required for caspofungin tolerance. Notably, the cell surface integrity sensor Wsc1p, but not the sensors Wsc2-4p and Mid2p, is required for sensing caspofungin perturbations. The expression modulation of PKC target genes requires the transcription factor Rlm1p, which controls expression of several cell wall synthesis and maintenance genes. Thus, caspofungin-induced cell wall damage requires Wsc1p as a dedicated sensor to launch a protective response through the activated salvage pathway for de novo cell wall synthesis. Our results establish caspofungin as a specific activator of Slt2p stress signaling in baker's yeast.

EAP1, a Candida albicans gene involved in binding human epithelial cells.

Abstract: Candida albicans adhesion to host tissues contributes to its virulence and adhesion to medical devices permits biofilm formation, but we know relatively little about the molecular mechanisms governing C. albicans adhesion to materials or mammalian cells. Saccharomyces cerevisiae provides an attractive model system for studying adhesion in yeast because of its well-characterized genetics and gene expression systems and the conservation of signal transduction pathways among the yeasts. In this study, we used a parallel plate flow chamber to screen and characterize attachment of a flo8Delta S. cerevisiae strain expressing a C. albicans genomic library to a polystyrene surface. The gene EAP1 was isolated as a putative cell wall adhesin. Sequence analysis of EAP1 shows that it contains a signal peptide, a glycosylphosphatidylinositol anchor site, and possesses homology to many other yeast genes encoding cell wall proteins. In addition to increasing adhesion to polystyrene, heterologous expression of EAP1 in S. cerevisiae and autonomous expression of EAP1 in a C. albicans efg1 homozygous null mutant significantly enhanced attachment to HEK293 kidney epithelial cells. EAP1 expression also restored invasive growth to haploid flo8Delta and flo11Delta strains as well as filamentous growth to diploid flo8/flo8 and flo11/flo11 strains. Transcription of EAP1 in C. albicans is regulated by the transcription factor Efg1p, suggesting that EAP1 expression is activated by the cyclic AMP-dependent protein kinase pathway.

Calcineurin Is Essential for Candida albicans Survival in Serum and Virulence

Abstract: Calcineurin is a calcium-activated protein phosphatase that is the target of the immunosuppressants cyclosporin A and FK506. In T cells, calcineurin controls nuclear import of the NF-AT transcription factor and gene activation. In plants and fungi, calcineurin functions in stress responses (e.g., temperature, cations, and pH) and is necessary for the virulence of the fungal pathogen Cryptococcus neoformans. Here we show that calcineurin is also required for the virulence of another major fungus that is pathogenic to humans, Candida albicans. C. albicans calcineurin mutants had significantly reduced virulence in a murine model of systemic infection. In contrast to its role in C. neoformans, calcineurin was not required for C. albicans survival at 37°C. Moreover, C. albicans calcineurin mutant strains exhibited no defects in known Candida virulence traits associated with host invasion, including filamentous growth, germ tube formation, and adherence to and injury of mammalian cells. C. albicans calcineurin mutant strains failed to colonize and grow in the kidneys of infected animals and were unable to survive when exposed to serum in vitro. Our studies illustrate that calcineurin has evolved to control aspects of the virulence of two divergent fungal pathogens via distinct mechanisms that can be targeted to achieve broad-spectrum antifungal action. Candida albicans is the most common cause of invasive fungal disease. Interestingly, C. albicans is also a component of the normal human biota that resides on mucosal surfaces, including the gastrointestinal tract and the oral and vaginal mucosae. Superficial colonization of immunocompetent or immunocompromised patients by the fungus can lead to oral thrush, vulvovaginitis, esophagitis, and cutaneous infections. Patients who have undergone abdominal surgery or organ transplantation, have late-onset diabetes, are taking broad-spectrum antibiotics, or are neutropenic are all at increased risk for serious invasive disease (6, 43, 58). Often, the first stage of invasive disseminated infection is candidemia, a bloodstream infection with Candida that can be associated with foreign-body catheters or entry of yeast through a damaged intestinal mucosa (reviewed in reference 7). Candidemia can progress to acute or chronic hematogenously disseminated candidiasis, in which organs such as the liver, kidney, and spleen are involved. During the progression from a common commensal organism to a pathogen, C. albicans faces a number of environments in vivo that require it to sense and respond to the extracellular milieu for survival. A great deal is known about the events leading to the pathogenic transition of C. albicans, especially about those pathways important for tissue invasion: the morphological switch from yeast-like growth to filamentous growth, expression of adherence molecules in the cell wall, and protease production (7, 11,

Chlamydomonas reinhardtii at the Crossroads of Genomics

Abstract: Simple, experimentally tractable systems such Saccharomyces cerevisiae, Chlamydomonas reinhardtii, and Arabidopsis thaliana are powerful models for dissecting basic biological processes. The unicellular green alga C. reinhardtii is amenable to a diversity of genetic and molecular manipulations. This haploid organism grows rapidly in axenic cultures, on both solid and liquid medium, with a sexual cycle that can be precisely controlled. Vegetative diploids are readily selected through the use of complementing auxotrophic markers and are useful for analyses of deleterious recessive alleles. These genetic features have permitted the generation and characterization of a wealth of mutants with lesions in structural, metabolic and regulatory genes. Another important feature of C. reinhardtii is that it has the capacity to grow with light as a sole energy source (photoautotrophic growth) or on acetate in the dark (heterotrophically), facilitating detailed examination of genes and proteins critical for photosynthetic or respiratory function. Other important topics being studied using C. reinhardtii, many of which have direct application to elucidation of protein function in animal cells (26), include flagellum structure and assembly, cell wall biogenesis, gametogenesis, mating, phototaxis, and adaptive responses to light and nutrient environments (32, 44). Some of these studies are directly relevant to applied problems in biology, including the production of clean, solar-generated energy in the form of H2, and bioremediation of heavy metal wastes. Recent years have seen the development of a molecular toolkit for C. reinhardtii (42, 44, 66, 98, 99). Selectable markers are available for nuclear and chloroplast transformation (4, 5, 12, 13, 30, 44, 56, 82). The Arg7 (22) and Nit1 (30) genes are routinely used to rescue recessive mutant phenotypes. The bacterial ble gene (which codes for zeocin resistance [70, 112]) is an easily scored marker for nuclear transformation, and the bacterial aadA gene (which codes for spectinomycin and streptomycin resistance) is a reliable marker for chloroplast transformation (39). Nuclear transformation can be achieved by

The Intestinal Protozoan Parasite Entamoeba histolytica Contains 20 Cysteine Protease Genes, of Which Only a Small Subset Is Expressed during In Vitro Cultivation

Abstract: Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.

Proteomics of Chlamydomonas reinhardtii Light-Harvesting Proteins

Abstract: With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of Chlamydomonas reinhardtii light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific lhcbm gene products. Interestingly, lhcbm6 gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different lhca-related gene products were predicted by comparison of the mass spectrometric data against Chlamydomonas expressed sequence tag and genomic databases, demonstrating the extensive variability of the C. reinhardtii Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in C. reinhardtii and will be an important basis of future functional studies addressing this diversity.

Metabolic-Flux Profiling of the Yeasts Saccharomyces cerevisiae and Pichia stipitis

Abstract: The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.

Expressed Sequence Tag Analysis of the Human Pathogen Paracoccidioides brasiliensis Yeast Phase: Identification of Putative Homologues of Candida albicans Virulence and Pathogenicity Genes

Abstract: Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5′ and 3′ ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.

Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress.

Abstract: In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.

Evidence of Sexual Recombination among Cryptococcus neoformans Serotype A Isolates in Sub-Saharan Africa

Abstract: The most common cause of fungal meningitis in humans, Cryptococcus neoformans serotype A, is a basidiomycetous yeast with a bipolar mating system. However, the vast majority (>99.9%) of C. neoformans serotype A isolates possess only one of the two mating type alleles (MATα). Isolates with the other allele (MATa) were recently discovered and proven to mate in the laboratory. It has been a mystery whether and where C. neoformans strains undergo sexual reproduction. Here, we applied population genetic approaches to demonstrate that a population of C. neoformans serotype A clinical isolates from Botswana contains an unprecedented proportion of fertile MATa isolates and exhibits evidence of both clonal expansion and recombination within two partially genetically isolated subgroups. Our findings provide evidence for sexual recombination among some populations of C. neoformans serotype A from sub-Saharan Africa, which may have a direct impact on their evolution.

Regulation of the Transcriptional Response to Oxidative Stress in Fungi: Similarities and Differences

Abstract: Fungal cells must deal with a wide variety of potentially toxic environmental challenges during the course of their proliferation. Often these environmental changes are designed to target and eliminate the fungus along with other microorganisms from a specific milieu, such as an animal host. An

Candida albicans response regulator gene SSK1 regulates a subset of genes whose functions are associated with cell wall biosynthesis and adaptation to oxidative stress.

Abstract: Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30 degrees C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.

Cell Biology of Mating in Candida albicans

Abstract: It was recently demonstrated that strains homozygous for either of the mating type-like loci MTLa and MTLα of Candida albicans undergo white-opaque switching and that expression of the opaque-phase phenotype greatly enhances mating between strains. Exploiting the latter property to obtain high-frequency mating, we have characterized the cell biology of the mating process of C. albicans. Employing continuous videomicroscopy, computer-assisted three-dimensional reconstruction of living cells, and fluorescence microscopy, we have monitored the mating-associated processes of conjugation, tube formation, fusion, budding, septum formation, and daughter cell development and the spatial and temporal dynamics of nuclear migration and division. From these observations, a model for the stages in C. albicans mating is formulated. The stages include shmooing, chemotropism of conjugation tubes, fusion of tubes and nuclear association, vacuole expansion and nuclear separation in the conjugation bridge, asynchronous nuclear division in the zygote, bud growth, nuclear migration into the daughter cell, septation, and daughter cell budding. Since there was no cytological indication of karyogamy, genetic experiments were performed to assess marker segregation. Recombination was not observed, suggesting that mating takes place in the absence of karyogamy between naturally occurring, homozygous a and α strains. This study provides the first description of the cell biology of the mating process of C. albicans.

Mating and Pathogenic Development of the Smut Fungus Ustilago maydis Are Regulated by One Mitogen-Activated Protein Kinase Cascade

Abstract: In the phytopathogenic fungus Ustilago maydis, pheromone-mediated cell fusion is a prerequisite for the generation of the infectious dikaryon. The pheromone signal elevates transcription of the pheromone genes and elicits formation of conjugation hyphae. Cyclic AMP and mitogen-activated protein kinase (MAPK) signaling are involved in this process. The MAPK cascade is presumed to be composed of Ubc4 (MAPK kinase kinase), Fuz7 (MAPK kinase), and Ubc3/Kpp2 (MAPK). We isolated the kpp4 gene and found it to be allelic to ubc4. Epistasis analyses with constitutively active alleles of kpp4 and fuz7 substantiate that Kpp4, Fuz7, and Kpp2/Ubc3 are components of the same module. Moreover, we demonstrate that Fuz7 activates Kpp2 and shows interactions in vitro. Signaling via this cascade regulates expression of pheromone-responsive genes, presumably through acting on the transcription factor Prf1. Interestingly, the same cascade is needed for conjugation tube formation, and this process does not involve Prf1. In addition, fuz7 as well as kpp4 deletion strains are nonpathogenic, while kpp2 deletion mutants are only attenuated in pathogenesis. Here we show that strains expressing the unphosphorylatable allele kpp2T182A/Y184F are severely affected in tumor induction and display defects in early infection-related differentiation.

TmkA, a Mitogen-Activated Protein Kinase of Trichoderma virens, Is Involved in Biocontrol Properties and Repression of Conidiation in the Dark

Abstract: Trichoderma virens is a mycoparasitic fungus used in biocontrol of soilborne plant pathogens. It inhibits or kills plant-pathogenic fungi through production of antifungal antibiotics and parasitism of hyphae and sclerotia. Conidiation, or the production of asexual spores, an inducible process triggered by light or nutrient stress, is an important trait in survival and also development of formulation products. In many fungi, signaling pathways, including mitogen-activated protein kinase (MAPK) cascades, have been implicated in parasitism of host plants as well as in the production of asexual spores. Here, we have studied the role of a MAPK gene, that for TmkA, in conidiation and antagonistic properties of a biocontrol strain of T. virens. Through single- and double-crossover recombination, we obtained three tmkA loss-of-function mutants. The TmkA transcript was not detectable in these mutants. The mutants conidiated in the dark, although photoinduction was normal and the light sensitivities of the wild type and the mutant were the same. The mutants had, overall, normal colony morphology, but their radial growth rate was reduced by about 16%, with no decrease in biomass production. Against Rhizoctonia solani hyphae, the knockout mutants exhibited mycoparasitic coiling and lysis of host hyphae similar to that of the wild type. The mutants, however, were less effective in colonizing the sclerotia of R. solani. On Sclerotium rolfsii, the MAPK loss-of-function mutants had reduced antagonistic properties in confrontation assays and failed to parasitize the sclerotia. TmkA-dependent and -independent pathways are thus involved in antagonism against different hosts. Finally, in contrast to the case for other filamentous fungi studied so far, signaling through a MAPK represses, rather than induces, asexual sporulation.

Molecular map of the Chlamydomonas reinhardtii nuclear genome

Abstract: We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.

Functional characterization of Candida albicans ABC transporter Cdr1p.

Abstract: In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).

Cryptococcus neoformans Gene Expression during Experimental Cryptococcal Meningitis

Abstract: Cryptococcus neoformans, an encapsulated basidiomycete fungus of medical importance, is capable of crossing the blood-brain barrier and causing meningitis in both immunocompetent and immunocompromised individuals. To gain insight into the adaptation of the fungus to the host central nervous system (CNS), serial analysis of gene expression (SAGE) was used to characterize the gene expression profile of C. neoformans cells recovered from the CNS of infected rabbits. A SAGE library was constructed, and 49,048 tags were sequenced; 16,207 of these tags were found to represent unique sequences or tag families. Of the 304 most-abundant tags, 164 were assigned to a putative gene for subsequent functional grouping. The results (as determined according to the number of tags that identified genes encoding proteins required for these functions) indicated that the C. neoformans cells were actively engaged in protein synthesis, protein degradation, stress response, small-molecule transport, and signaling. In addition, a high level of energy requirement of the fungal cells was suggested by a large number of tags that matched putative genes for energy production. Taken together, these findings provide the first insight into the transcriptional adaptation of C. neoformans to the host environment and identify the set of fungal genes most highly expressed during cerebrospinal fluid infection.