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At least 811 records · Page 45Linked to original sources
PubMed1991

Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans.

Cell surface hydrophobicity (CSH) of blastoconidia and blastoconidia bearing germ tubes of Candida albicans ATCC 26555 was monitored by assessing attachment of polystyrene microspheres to the cell surface, and we found that mature hyphae were significantly hydrophobic. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) or proteases abolished or significantly reduced attachment of latex beads to hyphae. This effect paralleled an obvious reduction in CSH of the entire cell population, as measured by an aqueous-hydrocarbon biphasic partitioning assay. Analysis of the cell wall material released by Zymolyase and adsorbed on polystyrene microspheres indicated that germ tube-specific cell wall proteins and mannoproteins with apparent molecular masses of 20 to 67 kDa may be responsible for the hydrophobicity of hyphae. Zymolyase released from blastoconidia cell walls a different set of proteins and mannoproteins that were able to adsorb to polystyrene microbeads. Such molecular species might in turn be responsible for the CSH exhibited by blastoconidium populations as determined by the biphasic partitioning assay, although these probably hydrophobic components can be masked on the surface of blastoconidia, as the latter had no or very few latex microspheres attached to their surfaces. Treatment of cells of both C. albicans morphologies with 2-mercaptoethanol released qualitatively distinct species of polystyrene-adsorbed proteins and mannoproteins from yeast and mycelial cells. These observations suggested that hydrophobic proteins and mannoproteins that could be associated with CSH are bound to the cell wall structure through diverse types of linkages.

Adsorption↗
PubMed1993

Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans.

The gene ECE1 (extent of cell elongation 1) was isolated by differential hybridization screening of a Candida albicans cDNA library by using probes derived from populations of yeast cells or hyphae. Expression of this gene was not detected when C. albicans grew as a budding yeast cell but was observed within 30 min after cells had been induced to form hyphae. In all strains tested, regardless of the induction signal, ECE1 expression correlated with the extent of cell elongation. The genomic version of ECE1 was cloned and sequenced. The deduced 271-amino-acid polypeptide consisted of eight tandem repeats of a degenerate 34-amino-acid sequence which contained no discernible homology with other known sequences. An ECE1 null mutant displayed no morphological alterations, which demonstrated that ECE1 is not essential for cell elongation or hypha formation despite the strict morphological association of its expression.

Amino Acid Sequence↗
PubMed1994

Differences in adhesion of Candida albicans 3153A cells exhibiting switch phenotypes to buccal epithelium and stratum corneum.

Cells of the laboratory strain 3153A of Candida albicans can be stimulated to undergo high-frequency phenotypic switching by a low dose of UV. We have compared the adhesive properties of cells exhibiting the basic original smooth (o-smooth) phenotype and three switch phenotypes (star, irregular wrinkle, and revertant smooth) to buccal epithelium and stratum corneum. The generalized hierarchy of adhesion is as follows: o-smooth > irregular wrinkle > revertant smooth > star. This is the inverse of the hierarchy of the proportions of elongate hyphae formed by these phenotypes in culture. These results suggest that the differences in adhesion between o-smooth and the three switch phenotypes of strain 3153A reflect, at least in part, the level of interference due to the formation of elongate hyphae, which tend to cause clumping in suspension. No major differences in the levels of adhesion of cells of the different phenotypes between buccal epithelium and stratum corneum were observed. Results which demonstrate that buccal epithelium induces germination (hypha formation) by conditioning the medium are also presented.

Adhesiveness↗
PubMed1996

Cloning and expression of the complement fixation antigen-chitinase of Coccidioides immitis.

A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen. In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase. The PCR product was used as a hybridization probe to screen a developing spherule-(lambda)ZAP cDNA library, and three hybridizing clones were selected. These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins. Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen. The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa. When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C. immitis CF-chitinase. Nondenaturing gels of the pCTS 4-2A E. coli lysates and the purified C. immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities. The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae. Thus, a functional recombinant CF-chitinase antigen was produced in E. coli and was used in serological diagnostic applications. These results also suggest a functional role for this chitinase in SE development and maturation.

Amino Acid Sequence↗
PubMed2001

Differential expression of the Aspergillus fumigatus pksP gene detected in vitro and in vivo with green fluorescent protein.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To be able to analyze the expression of putative virulence-associated genes of A. fumigatus, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5' sequences, containing the putative promoters of the pyrG gene, encoding orotidine-5'-phosphate decarboxylase, and the pksP gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence of A. fumigatus, were fused with the egfp gene. The PpksP-egfp construct was integrated via homologous recombination into the genomic pksP locus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatus was observed. Fluorescence derived from the PYRG-EGFP fusion protein was detected during all developmental stages of the fungus, i.e., during germination, during vegetative growth, in conidiophores, and weakly in conidia. In addition, it was also detected in germinating conidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidiophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly, pksP-egfp expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that the pksP gene can also be expressed in hyphae under certain conditions and, furthermore, that the pksP gene might also contribute to invasive growth of the fungus.

Amino Acid Sequence↗
PubMed2002

Lymphocyte adhesion to Candida albicans.

Adherence of lymphocytes to the fungus is the first step in the direct lymphocyte-mediated antifungal effect against Candida albicans. In this study we identified macrophage-1 antigen (Mac-1) (CD11b/CD18, alpha(M)/beta(2)) as the lymphocyte surface structure responsible for the adhesion of activated lymphocytes to the hyphal form of the fungus. Antibodies specific for epitopes of the alpha-subunit (CD11b) and the beta(2)-subunit (CD18) of Mac-1 were shown to completely eliminate lymphocyte adhesion to C. albicans hyphae. Lymphocyte adhesion to C. albicans was also inhibited significantly by known ligands of Mac-1, including the extracellular matrix proteins laminin and fibrinogen, as well as engineered peptides containing arginine-glycine-aspartic acid sequences and the disintegrin echistatin. N-Acetyl-D-glucosamine and beta-glucan, which inhibit Mac-1-mediated adhesion to the yeast, blocked lymphocyte adhesion to hyphae. NIH 3T3 fibroblast transfectants expressing human CD11b/CD18 bound to C. albicans, and their binding was inhibited by antibodies specific for CD11b/CD18. Finally, antibodies specific for CD11b/CD18 effectively inhibited the capacity of activated lymphocytes to have an antifungal effect against hyphae. Our results clearly identify Mac-1 (CD11b/CD18) as the lymphocyte surface structure that mediates activated lymphocyte adhesion to C. albicans and the resultant antifungal effect of the lymphocytes.

3T3 Cells↗
PubMed2006

Interactions between Streptomyces coelicolor and Bacillus subtilis: Role of surfactants in raising aerial structures.

Using mixed-species cultures, we have undertaken a study of interactions between two common spore-forming soil bacteria, Bacillus subtilis and Streptomyces coelicolor. Our experiments demonstrate that the development of aerial hyphae and spores by S. coelicolor is inhibited by surfactin, a lipopeptide surfactant produced by B. subtilis. Current models of aerial development by sporulating bacteria and fungi postulate a role for surfactants in reducing surface tension at air-liquid interfaces, thereby removing the major barrier to aerial growth. S. coelicolor produces SapB, an amphipathic peptide that is surface active and required for aerial growth on certain media. Loss of aerial hyphae in developmental mutants can be rescued by addition of purified SapB. While a surfactant from a fungus can substitute for SapB in a mutant that lacks aerial hyphae, not all surfactants have this effect. We show that surfactin is required for formation of aerial structures on the surface of B. subtilis colonies. However, in contrast to this positive role, our experiments reveal that surfactin acts antagonistically by arresting S. coelicolor aerial development and causing altered expression of developmental genes. Our observations support the idea that surfactants function specifically for a given organism regardless of their shared ability to reduce surface tension. Production of surfactants with antagonistic activity could provide a powerful competitive advantage during surface colonization and in competition for resources.

Bacillus subtilis↗
PubMed1970

Fatty acid toxicity and methyl ketone production in Aspergillus niger.

Vegetative hyphae of Aspergillus niger rapidly converted caproic acid into 2-pentanone. More caproic acid was required for maximal ketone production at alkaline as compared to acidic pH values. Further increases in caproate concentrations at each pH value tested (4.5, 5.5, 6.5, 7.5, and 8.5) resulted in inhibition of ketone production and O(2) uptake. At alkaline pH values (8.5 and 7.5), oxygen uptake above the endogenous level and the production of 2-pentanone were parallel. This relationship did not hold at acidic pH values. At these pH values, ketone production continued (pH 6.5) or attained a maximum (pH 5.5 and 4.5) at caproate concentrations at which oxygen uptake was inhibited below endogenous levels. These data indicate that endogenous oxygen uptake was not inhibited by caproate at alkaline pH values at concentrations which did inhibit caproate oxidation and 2-pentanone production. Conversely, at acidic pH values, endogenous oxygen uptake was vigorously inhibited by caproate at concentrations at which exogenous fatty acid oxidation and 2-pentanone production were less affected. Simon-Beevers plots of these data showed that the undissociated acid was the permeant form of caproic acid. The fatty anion appeared to be the active or inhibitory form of caproate within the cell. Vegetative hyphae of A. niger were poorly buffered. Once the hyphae were washed and resuspended in phosphate buffer, they were well buffered towards inhibitory concentrations of caproic acid. These findings suggest that the primary mechanism(s) by which caproate inhibits oxygen uptake and ketone formation does not involve a change in the intracellular pH.

Aspergillus↗
PubMed1970

Biochemical and histochemical localization of invertase in Neurospora crassa during conidial germination and hyphal growth.

The intracellular localization of Neurospora invertase, an enzyme partially secreted and partially retained by Neurospora at the cell periphery, was investigated. A cell wall fraction was isolated, to which 24% of the cell-bound invertase was firmly attached. A sensitive osmiophilic stain for invertase was developed and used in conjunction with the technique of indirect immunofluorescence to follow the pattern of invertase localization during the development of Neurospora from the germination of conidia to the mature hypha. These studies revealed that: (i) conidial invertase was uniformly distributed along the cell periphery; (ii) growing hyphal tips of germinating conidia showed pronounced invertase activity as the rest of the conidial cell wall lost its peripheral activity; (iii) hyphae in early log-phase growth had strong enzyme activity associated with the cell wall, and in late log phase the activity became associated with the plasma membrane and points where new hyphal branches were being formed; and (iv) hyphae in early stationary phase had strong fluorescence at incipient branching points, in "dots" close to the plasma membrane, and in the cytoplasm.

Animals↗
PubMed1973

Budding in the dimorphic fungus Phialophora dermatitidis.

Ultrastructural comparisons of yeast and hyphal bud formation in Phialophora dermatitidis reveal that bud initiation is characterized by a blastic rupture of the outer portion of the yeast or hyphal wall and the emergence of a bud protuberance through the resulting opening. The wall of the emerging bud is continuous, with only an inner wall layer of the parental yeast or hypha. The outer, ruptured portion of the parental wall typically forms a collar around the constricted emergence region of the developing bud. The cytoplasm within the very young emerging bud invariably contains a small number of membrane-bound vesicles. The septum formed between the daughter bud and the parental yeast or hypha is a complete septum devoid of a septal pore, septal pore plug, or any associated Woronin bodies characteristic of simple septa of the moniliform or true hyphae. These observations suggest that yeast bud formation and lateral hyphal bud formation in the dimorphic fungus P. dermatitidis involve a growth process which occurs identically in both the yeast and mold phase of this human pathogenic organism.

Cell Wall↗
PubMed1973

Nuclear apparatus of Hyphomicrobium.

The nuclear apparatus of Hyphomicrobium sp. strain B-522 is examined by various microscopy and radiolabeling techniques to determine its behavior during the reproductive cycle of these bacteria. The young, swarmer cell contains a single nucleoid comprised of a deoxyribonucleic acid (DNA) molecule with a molecular weight of 3.1 x 10(9). After development of the swarmer into a mature mother cell with a hypha and bud, the nucleoid replicates and separates into two daughter nucleoids during the initial stages of bud formation. After further development of the bud, one of the daughter nucleoids in the mother cell is rapidly transferred through the hypha to the bud. Half of the old DNA strands pass to each consecutive generation of daughter cells, but only 43% of the stable ribonucleic acid is transferred. The role which the hypha plays in the developmental cycle of these bacteria is discussed, and a mechanism for nuclear transfer is proposed.

Adenine↗
PubMed1974

Morphogenesis and ultrastructure of Claviceps purpurea during submerged alkaloid formation.

Criteria for morphogenetic and ultrastructural distinction between conidia and chlamydospores of a submerged culture of Claviceps purpurea (Fr.) Tul. are described. Both the hyphae of the sphacelia (asexual) stage and the conidia contained granular cytoplasm. Cytoplasmic invaginations in vacuoles were transformed to electron-opaque bodies and disintegrated prior to germination. The budding of conidia had basipetal succession. The chlamydospores were formed by rounding up the terminal cells of filamentous hyphae. Homogeneous nonvacuolized cytoplasm with lipid droplets and lipid-forming bodies was characteristic of young chlamydospores. Cristate mitochondria did not appear in the chlamydospores before the alkaloid production phase. Simultaneously a specific organelle in the chlamydospores, a dense body, appeared to absorb intracellular lipids and form large deposits of phospholipid material. No germination of chlamydospores was observed. The ultrastructural pattern described for chlamydospores was also observed in hyphae with reduced proliferation during the alkaloid production phase.

Cell Membrane↗
PubMed1982

Production and composition of an exocellular Nigeran-protein complex isolated from cultures of Aspergillus awamori.

Aspergillus awamori Nakazawa (QM873) hyphae maintained on a nitrogen-deficient medium produced an exocellular complex consisting of the wall alpha-glucan, nigeran (94%), water-soluble nigeran oligosacchrides (1 to 2%), protein (2 to 4%), and a small amount of beta-glycan (less than 1%). Nigeran was not covalently linked to protein. The complex appeared in the growth medium only under those temporal or nutritional conditions where the hyphal wall content to nigeran reached at least 30% of the cell dry weight. Rates of nigeran accumulation in the hypha cell wall, scanning electron microscopy of hyphae, and pulse-chase experiments with [14C]glucose suggested that the complex arises via displacement of a portion of the hyphal wall into the medium. The nigeran portion of the complex contained lamellar crystalline domains similar to those in the intact cell wall. Enzymic digestion of nigeran in the complex indicated that it has a degree of crystallinity lower than that observed with pure nigeran crystals grown in vitro. Therefore, polymerization in vivo resulted in somewhat less chain organization in the crystallite. Since this complex was isolated without any prior chemical or exogenous enzymatic treatment, it should be useful for studies of hyphal wall biogenesis and organization in this organism.

Aspergillus↗
PubMed1999

Ras signaling is required for serum-induced hyphal differentiation in Candida albicans.

Serum induces Candida albicans to make a rapid morphological change from the yeast cell form to hyphae. Contrary to the previous reports, we found that serum albumin does not play a critical role in this morphological change. Instead, a filtrate (molecular mass, <1 kDa) devoid of serum albumin induces hyphae. To study genes controlling this response, we have isolated the RAS1 gene from C. albicans by complementation. The Candida Ras1 protein, like Ras1 and Ras2 of Saccharomyces cerevisiae, has a long C-terminal extension. Although RAS1 appears to be the only RAS gene present in the C. albicans genome, strains homozygous for a deletion of RAS1 (ras1-2/ras1-3) are viable. The Candida ras1-2/ras1-3 mutant fails to form germ tubes and hyphae in response to serum or to a serum filtrate but does form pseudohyphae. Moreover, strains expressing the dominant active RAS1(V13) allele manifest enhanced hyphal growth, whereas those expressing a dominant negative RAS1(A16) allele show reduced hyphal growth. These data show that low-molecular-weight molecules in serum induce hyphal differentiation in C. albicans through a Ras-mediated signal transduction pathway.

Amino Acid Sequence↗
PubMed2002

ATP-binding cassette transport system involved in regulation of morphological differentiation in response to glucose in Streptomyces griseus.

Streptomyces griseus NP4, which was derived by UV mutagenesis from strain IFO13350, showed a bald and wrinkled colony morphology in response to glucose. Mutant NP4 formed ectopic septa at intervals along substrate hyphae, and each of the compartments developed into a spore which was indistinguishable from an aerial spore in size, shape, and thickness of the spore wall and in susceptibility to lysozyme and heat. The ectopic spores of NP4 formed in liquid medium differed from "submerged spores" in lysozyme sensitivity. Shotgun cloning experiments with a library of the chromosomal DNA of the parental strain and mutant NP4 as the host gave rise to DNA fragments giving two different phenotypes; one complementing the bald phenotype of the host, and the other causing much severe wrinkled morphology in the host. Subcloning identified a gene (dasR) encoding a transcriptional repressor belonging to the GntR family that was responsible for the reversal of the bald phenotype and a gene (dasA) encoding a lipoprotein probably serving as a substrate-binding protein in an ATP-binding cassette (ABC) transport system that was responsible for the severe wrinkled morphology. These genes were adjacent but divergently encoded. Two genes, named dasB and dasC, encoding a membrane-spanning protein were present downstream of dasA, which suggested that dasRABC comprises a gene cluster for an ABC transporter, probably for sugar import. dasR was transcribed actively during vegetative growth, and dasA was transcribed just after commencement of aerial hypha formation and during sporulation, indicating that both were developmentally regulated. Transcriptional analysis and direct sequencing of dasRA in mutant NP4 suggested a defect of this mutant in the regulatory system to control the expression of these genes. Introduction of multicopies of dasA into the wild-type strain caused ectopic septation in very young substrate hyphae after only 1 day of growth and subsequent sporulation in response to glucose. The ectopic spores of the wild type had a thinner wall than those of mutant NP4, in agreement with the observation that the former was sensitive to lysozyme and heat. Disruption of the chromosomal dasA or dasR in the wild-type strain resulted in growth as substrate mycelium, suggesting an additional role of these genes in aerial mycelium formation. The ectopic septation and sporulation in mutant NP4 and the wild-type strain carrying multicopies of dasA were independent of a microbial hormone, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), that acts as a master switch of aerial mycelium formation and secondary metabolism.

ATP-Binding Cassette Transporters↗
PubMed2002

Control by A-factor of a metalloendopeptidase gene involved in aerial mycelium formation in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis. An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner. A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA-binding sites. The primary product of sgmA consisted of N-terminal pre, N-terminal pro, mature, and C-terminal pro regions. sgmA was transcribed in an AdpA-dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation. AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position -60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position -260 (B site), as determined by DNase I footprinting. Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation. Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day. SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation.

4-Butyrolactone↗
PubMed2005

Dynamics of FtsZ assembly during sporulation in Streptomyces coelicolor A3(2).

FtsZ, the bacterial tubulin homologue, is the main player in at least two distinct processes of cell division during the development of Streptomyces coelicolor A3(2). It forms cytokinetic rings and is required for the formation of both the widely spaced hyphal cross walls in the substrate mycelium and the specialized septation that converts sporogenic aerial hyphae into spores. The latter developmentally controlled septation involves the coordinated assembly of large numbers of FtsZ rings in each sporulating hyphal cell. We used an FtsZ-enhanced green fluorescent protein (EGFP) translational fusion to visualize the progression of FtsZ ring assembly in vivo during sporulation of aerial hyphae. This revealed that the regular placement of multiple FtsZ rings and initiation of cytokinesis was preceded by a protracted phase during which spiral-shaped FtsZ intermediates were detected along the length of the aerial hyphal cell. Time course experiments indicated that they were remodeled and gradually replaced by regularly spaced FtsZ rings. Such spiral-shaped filaments could also be detected with immunofluorescence microscopy using an antiserum against FtsZ. Based on our observations, we propose a model for the progression of Z-ring assembly during sporulation of S. coelicolor. Furthermore, mutants lacking the developmental regulatory genes whiA, whiB, whiG, whiH, and whiI were investigated. They failed in up-regulation of the expression of FtsZ-EGFP in aerial hyphae, which is consistent with the known effects of these genes on ftsZ transcription.

Bacterial Proteins↗
PubMed2006

Influence of CrgA on assembly of the cell division protein FtsZ during development of Streptomyces coelicolor.

The product of the crgA gene of Streptomyces coelicolor represents a novel family of small proteins. A single orthologous gene is located close to the origin of replication of all fully sequenced actinomycete genomes and borders a conserved gene cluster implicated in cell growth and division. In S. coelicolor, CrgA is important for coordinating growth and cell division in sporogenic hyphae. In this study, we demonstrate that CrgA is an integral membrane protein whose peak expression is coordinated with the onset of development of aerial hyphae. The protein localizes to discrete foci away from growing hyphal tips. Upon overexpression, CrgA localizes to apical syncytial cells of aerial hyphae and inhibits the formation of productive cytokinetic rings of the bacterial tubulin homolog FtsZ, leading to proteolytic turnover of this major cell division determinant. In the absence of known prokaryotic cell division inhibitors in actinomycetes, CrgA may have an important conserved function influencing Z-ring formation in these bacteria.

Amino Acid Sequence↗