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PubMed2001

Inhibition of hyphal growth of Candida albicans by activated lansoprazole, a novel benzimidazole proton pump inhibitor.

The effect of activated lansoprazole (AG 2000), a novel benzimidazole proton pump inhibitor, against hypha formation of Candida albicans was examined in hypha-forming medium pH 7 (HFM7) after 20 h. AG 2000, at 50-800 microM, did not inhibit germ tube formation. However, it inhibited elongation of germ tubes to form hyphae and favored conversion of germ tubes to resume yeast growth at concentrations of > or =200 microM. Pre-treatment of AG 2000 with a sulfhydryl reagent (1:1), such as 2-mercaptoethanol. blocked the inhibitory property of AG 2000 on hypha formation.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2002

Unusual presentation of Microsporum canis in human hair.

This study reports an unusual presentation of Microsporum canis in infected human hair in a three-year-old boy. Infected hairs showed ectothrix colonization with a mass of small conidia and numerous macroconidia typical of the genus Microsporum. The infected hairs also showed strong invasion by hyphae bearing small numbers of arthroconidia. These hyphae were thinner and longer than those normally found in hair and scalp. It is well known that in cases of tinea capitis due to M. canis, ectothrix arthroconidia formation is extrapilary and arises from intrapilary hyphae. In this case it seems that unusual quantities of hyphae were formed, and not all of them broke down into arthroconidia. They appeared to remain in the hair and to produce macroconidia, which were not formed outside the hair in contact with air, but instead seemed to remain in a deeper layer of the hair than the arthroconidia seen.

Child↗
PubMed2003

An investigation into the involvement of defense signaling pathways in components of the nonhost resistance of Arabidopsis thaliana to rust fungi also reveals a model system for studying rust fungal compatibility.

Seventeen accessions of Arabidopsis thaliana inoculated with the cowpea rust fungus Uromyces vignae exhibited a variety of expressions of nonhost resistance, although infection hypha growth typically ceased before the formation of the first haustorium, except in Ws-0. Compared with wild-type plants, there was no increased fungal growth in ndr1 or eds1 mutants defective in two of the signal cascades regulated by the major class of Arabidopsis host resistance genes. However, in the Col-0 background, infection hyphae of U. vignae and two other rust fungi were longer in sid2 mutants defective in an enzyme that synthesizes salicylic acid (SA), in npr1 mutants deficient in a regulator of the expression of SA-dependent pathogenesis related (PR) genes, and in NahG plants containing a bacterial salicylate hydroxylase. Infection hyphae of U. vignae and U. appendiculatus but not of Puccinia helianthi were also longer in jar1 mutants, which are defective in the jasmonic acid defense signaling pathway. Nevertheless, haustorium formation increased only for the Uromyces spp. and only in sid2 mutants or NahG plants. Rather than the hypersensitive cell death that usually accompanies haustorium formation in nonhost plants, Arabidopsis typically encased haustoria in calloselike material. Growing fungal colonies of both Uromyces spp., indicative of a successful biotrophic relationship between plant and fungus, formed in NahG plants, but only U. vignae formed growing colonies in the sid2 mutants and cycloheximide-treated wild-type plants. Growing colonies did not develop in NahG tobacco or tomato plants. These data suggest that nonhost resistance of Arabidopsis to rust fungi primarily involves the restriction of infection hypha growth as a result of defense gene expression. However, there is a subsequent involvement of SA but not SA-dependent PR genes in preventing the Uromyces spp. from forming the first haustorium and establishing a sufficient biotrophic relationship to support further fungal growth. The U. vignae-Arabidopsis combination could allow the application of the powerful genetic capabilities of this model plant to the study of compatibility as well as nonhost resistance to rust fungi.

Arabidopsis↗
PubMed1979

A model for hyphal growth and branching.

A mathematical model for hyphal growth and branching is described which relates cytological events within hyphae to mycelial growth kinetics. Essentially the model quantifies qualitative theories of hyphal growth in which it is proposed that vesicles containing wall precursors and/or enzymes required for wall synthesis are generated at a constant rate throughout a mycelium and travel to the tips of hyphae where they fuse with the plasma membrane, liberating their contents into the wall and increasing the surface area of the hypha to give elongation. The hypothesis that there is a duplication cycle in hyphae which is equivalent to the cell cycle observed in unicellular micro-organisms is also included in the model. Predictions from the model are compared with experimentally observed growth kinetics of mycelia of Geotrichum candidum and Aspergillus nidulans. The finite difference model which was constructed is capable of predicting changes in hyphal length and in the number and positions of branches and septa on the basis of changes in vesicle and nuclear concentration. Predictions were obtained using the model which were in good agreement with experimentally observed data.

Aspergillus nidulans↗
PubMed1989

Chemotropism and branching as alternative responses of Achlya bisexualis to amino acids.

Hyphae of Achlya bisexualis growing on lean media orient their extension towards a source of amino acids, and also put forth branches. Micropipettes were used to generate gradients of amino acids in the vicinity of individual hyphae. Phenylalanine and methionine were the most powerful attractants: 0.04 mM amino acid in the pipette produced reorientation, and higher concentrations made the hyphae curl around the pipette and grow into its tip. Hyphae detected gradients as low as 5% across their width. Methionine and phenylalanine appeared to bind to different receptors. Local application of these amino acids also elicited the emergence of single branches, next to the pipette and on the high side of the gradient; comparison of diverse amino acids and their analogues suggested that branching and chemotropism share common receptors. By contrast, cytochalasin A and various ionophores induced branches at random sites, without receptor involvement. We propose that binding of amino acids to their receptors determines the site of precursor vesicle exocytosis, and consider possible mechanisms.

Amino Acids↗
PubMed1991

Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora.

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmission electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCl), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6%) and proteins (19%). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.

Animals↗
PubMed1999

Candida albicans and Yarrowia lipolytica as alternative models for analysing budding patterns and germ tube formation in dimorphic fungi.

The site for bud selection and germ tube emission in two yeasts, Candida albicans and Yarrowia lipolytica, was analysed. Both dimorphic organisms display different patterns of budding, which also differ from those described for Saccharomyces cerevisiae. C. albicans, which is diploid and (until now) lacks a known sexual cycle, buds in an axial budding pattern. During the yeast-hypha transition induced by pH, serum, N-acetylglucosamine (GlcNAc) or temperature, germ tube emergence occurs at approximately 50% in a polar manner, while the other 50% of cells show non-polar germ tube emission. Y. lipolytica, in which most of the natural isolates are haploid and which has a well characterized sexual cycle, buds with a polar budding pattern independently of the degree of ploidy. Germ tube emission during the yeast-hypha transition in both haploid and diploid cells generally occurs at the pole distal from the division site (bipolar). The addition of hydroxyurea (HU), an inhibitor of DNA synthesis, also produces different effects. In its presence, and therefore in the absence of DNA synthesis, the yeast-hypha transition is completely abolished in Y. lipolytica. By contrast, in C. albicans germ tube emission in the presence of HU is similar to that observed in control cultures for at least 90 min under induction conditions. These results demonstrate that, rather than a single developmental model, several models of development should be invoked to account for the processes involved in the morphological switch in yeasts (the yeast-hypha transition).

Candida albicans↗
PubMed2000

Heterogeneous distribution of lysine 6-aminotransferase during cephamycin C biosynthesis in Streptomyces clavuligerus demonstrated using green fluorescent protein as a reporter.

The cellular distribution of the cephamycin biosynthetic enzyme lysine 6-aminotransferase (LAT) has been studied in Streptomyces clavuligerus hyphae by confocal microscopy using the S65T mutant of green fluorescent protein (GFP) as a reporter. LAT mediates the first committed step in the biosynthesis of the secondary metabolite cephamycin C by S. clavuligerus. The enzymic activity of LAT varies with time during the growth of S. clavuligerus in liquid medium. To investigate if this temporal variation occurs uniformly amongst all hyphae, S. clavuligerus was transformed with a plasmid containing the LAT-encoding gene translationally fused to the GFP-encoding gene. The LAT-GFP fusion product displayed fluorescence spectral characteristics of GFP, and showed similar temporal characteristics of LAT activity compared to the wild-type strain of S. clavuligerus. The transformed strain exhibited a heterogeneous distribution of fluorescence in mycelia grown in liquid cultures. This distribution varied significantly as the batch progressed: only a fraction of the mycelia fluoresced in the early growth phase, whereas nearly all hyphae fluoresced by the late growth phase. Thereafter, a non-uniform distribution of fluorescence was again observed in the declining growth phase. A large fraction of the non-fluorescent cells in the declining growth phase were found to be non-viable. Observations of S. clavuligerus colonies grown on solid agar also showed variation of LAT-GFP expression at different stages of growth. These observations in the solid phase can be explained in terms of nutrient deprivation and signalling molecules. The results suggest that physiological differentiation of S. clavuligerus mycelia leading to cephamycin C biosynthesis is both temporally and spatially distributed. The findings also revealed that the observed heterogeneity was independent of the position of individual cell compartments within the hypha. The potential of GFP as a reporter for the quantitative study of cephamycin biosynthesis at the cellular level has also been demonstrated.

Amino Acid Sequence↗
PubMed1976

Wall structure of the Neurospora hyphal apex: immunofluorescent localization of wall surface antigens.

Antisera have been raised in rabbits against three wall fractions from Neurospora crassa. Fractions were separated according to Mahadevan & Tatum (1965), i.e. fraction I, glucan-peptide-galactosamine complex; fraction III, laminarin-like glucan; and fraction IV, chitin. Distinct patterns of immunofluorescent staining were obtained using an indirect staining method. Hyphae stained with antiserum to fraction I showed maximum fluorescence in the apical and/or subapical regions: in both cases, fluorescence showed a sharp decrease with distance behing the subapical region. Hyphae stained with antiserum to fraction III showed faintly fluorescent tips with fluorescence increasing with distance from the tip. Hyphae stained with antiserum to fraction IV showed faint fluorescence, equivalent to levels of autofluorescence, except at the sites of hyphal fractures. Antisera were also raised against whole walls from 24 and 120 h cultures. Hyphae stained with antisera against whole walls which had previously been absorbed to remove antibodies to fractions I, III, and IV showed preferential staining of apices. The uncharacterized tip antigen(s) thus revealed was also demonstrated on immunodiffusion plates. This pattern of immunofluorescence was compared to the fluorescence of apices after staining with an optical brightener. Enzymic dissection procedures did not generally give reliable results with apices from 24 h cultures. Untreated apices appeared amorphous, while a drastic chemical treatment revealed randomly oriented microfibrils which were shown to be alpha-chitin. The apical hyphal walls were significantly thinner than those from more mature hyphal regions.

Antigens, Fungal↗
PubMed1976

Mitosis, septation, branching and the duplication cycle in Aspergillus nidulans.

Mitosis, septation and branching were studied in undifferentiated mycelia and leading hyphae of Aspergillus nidulans, a mould which forms incomplete septa. After spore germination, nuclei divided synchronously until germ-tube hyphae contained 8 or 16 nuclei; mitosis occurred when the volume of cytoplasm per haploid nucleus was about 60 mum3. Intercompartment development was not synchronized, consequently mitosis in the mycelium as a whole eventually became asynchronous. During the stage of asynchronous compartment development, the nuclei, septa, branches and total length of undifferentiated mycelia all increased exponentially at approximately the same specific rate. Septa were formed in hyphae in groups of up to nine; the mean time required for the formation of a group of septa was about 9 min. The mean interval between successive cycles of septation in a hypha was approximately the same as the organism's doubling time. There was a highly significant correlation coefficient between septation and branch initiation and most intercalary compartments initially formed a single branch. The volume of cytoplasm per nucleus in a diploid strain was approximately double the value observed for a haploid strain. However, the length of the hyphal growth unit was not affected by ploidy. The study suggests that a duplication cycle can be recognized during mycelial growth which is analogous to the cell cycle observed in unicellular micro-organisms.

Aspergillus nidulans↗
PubMed1998

Intracellular pH in arbuscular mycorrhizal fungi. A symbiotic physiological marker

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0-2 &mgr;m) was typically 6.7, increased sharply to 7.0 behind this region (9.5 &mgr;m), and decreased over the next 250 &mgr;m to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.

Journal Article↗
PubMed1992

Experimental dermatophilosis.

The histopathologic features of an experimental dermatophilosis of rabbit skin were described. The strain of Dermatophilus congolensis used for the experimental infection was obtained from the unique isolation and cultivation of this actinomycete from the ulcerous leg lesion of one male patient. Further strains in experiment came from the collections of type cultures. The experimental infection was characterized as an exudative and crustal dermatitis with acanthosis and subcorneal abscesses. Branching hyphae and clusters of coccoid bodies were found mostly in the parakeratotic layer and infrequently in the abscesses. Both the coccoid bodies and the hyphae were Gram-positive. The hyphae were acid-fast but the coccoid bodies were non-acid-fast. The fine structure of hyphae and coccoid bodies, namely of those with flagella, was also described by means of a transmission and scanning electron microscope.

Actinomycetales↗
PubMed2000

The chsA gene from Aspergillus nidulans is necessary for maximal conidiation.

A fragment from the open reading frame of the cloned chsA gene from Aspergillus nidulans was deleted and replaced with the argB gene. The resulting construct was used to replace the wild-type chsA gene in an argB deletion strain. The growth and morphology of the vegetative hyphae from the resulting chsA disruptant strain were indistinguishable from those of a wild-type strain but the chitin content of the hyphae from the disruptant was reduced to approximately 90% of that of wild-type. The disruptant showed reduced ability to produce the asexual spores (conidia) that are formed by differentiated aerial hyphae called conidiophores. The ability to form undifferentiated aerial hyphae was not impaired in the disruptant. The conidiophores and conidia produced by the disruptant were indistinguishable from those of wild-type. Conidium formation by the disruptant grown on a variety of media was reduced to about 30% of the wild-type. A chsE null strain did not show a defect in conidiation but a strain in which both chsA and chsE were inactivated produced about 3% of the conidia of wild-type. That finding supports the hypothesis that chsA and chsE encode a partially redundant function necessary for conidiophore development.

Aspergillus nidulans↗
PubMed1995

Association between carriage of oral yeasts and malnutrition among Tanzanian infants aged 6-24 months.

OBJECTIVE: To determine if there is an association between carriage of oral yeasts and malnutrition in infants. DESIGN: A case-control study within a cross-sectional study. The dependent variable was carriage of oral yeasts. The exposure variable was malnutrition and confounders to be adjusted for were age, sex, and breast-feeding. SETTING: A maternal and child health clinic in Dar-es-Salaam, Tanzania that offers routine medical check-ups to all expectant mothers and children aged between 0 and 5 years in its catchment areas. SUBJECTS AND METHODS: 972 infants aged 6-24 months participated. Smears from the tongue and cheek mucosa were examined for candidal hyphae and blastospores. Malnutrition was categorized according to Tanzanian standards (weight-for-age) and World Health Organization (WHO) standards (weight-for-height and height-for-age). MAIN OUTCOME MEASURE: Carriage of oral yeasts (hyphae and blastospores). RESULTS: Carriage of oral yeasts was significantly higher in the 227 malnourished compared with the 745 well nourished adjusted for confounders. Odds ratio for presence of hyphae in smears from the severely malnourished (weight-for-age) was 4.5 (90% CI: 2.0-10.0). Odds ratio for presence of hyphae was 2.3 (90% CI: 1.1-4.8) when weight-for-height were used to categorize for malnutrition. CONCLUSION: The study tends to confirm the generally held view that malnutrition may predispose to carriage of oral yeasts and subsequent oral candidiasis.

Candida↗
PubMed1998

Inhibition of hyphal growth of azole-resistant strains of Candida albicans by triazole antifungal agents in the presence of lactoferrin-related compounds.

The effects of bovine lactoferrin (LF) or the LF-derived antimicrobial peptide lactoferricin B (LFcin B) on the growth of Candida albicans hyphae, including those of three azole-resistant strains, were investigated by a crystal violet staining method. The hyphae of two highly azole-resistant strains were more susceptible to inhibition by LF or LFcin B than the azole-susceptible strains tested. One moderately azole-resistant strain was defective in the formation of hyphae and showed a susceptibility to LF greater than that of the susceptible strains but a susceptibility to LFcin B similar to that of the susceptible strains. The highly azole-resistant strain TIMM3317 showed trailing growth in the presence of fluconazole or itraconazole, while the extent of growth was reduced by the addition of LF or LFcin B at a sub-MIC. Thus, the addition of LF or LFcin B at a sub-MIC resulted in a substantial decrease in the MICs of fluconazole and itraconazole for two highly azole-resistant strains; e.g., the MIC of fluconazole for TIMM3317 was shifted from > 256 to 0.25 micrograms/ml by LF, but the MICs were not decreased for the susceptible strains. The combination effects observed with triazoles and LF-related compounds in the case of the two highly azole-resistant strains were confirmed to be synergistic by the fractional inhibitory concentration index. These results demonstrate that for some azole-resistant C. albicans strains, LF-related compounds combined with triazoles can inhibit the growth of hyphae, an important form of this organism in pathogenesis.

Antifungal Agents↗
PubMed2002

Exploration of inorganic C and N assimilation by soil microbes with time-of-flight secondary ion mass spectrometry.

Stable C and N isotopes have long been used to examine properties of various C and N cycling processes in soils. Unfortunately, relatively large sample sizes are needed for accurate gas phase isotope ratio mass spectrometric analysis. This limitation has prevented researchers from addressing C and N cycling issues on microbially meaningful scales. Here we explored the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to detect 13C and 15N assimilation by individual bacterial cells and to quantify N isotope ratios in bacterial samples and individual fungal hyphae. This was accomplished by measuring the relative abundances of mass 26 (12C14N-) and mass 27 (13C14N- and 12C15N-) ions sputtered with a Ga+ probe from cells adhered to an Si contact slide. TOF-SIMS was successfully used to locate and quantify the relative 15N contents of individual hyphae that grew onto Si contact slides in intimate contact with a model organomineral porous matrix composed of kaolin, straw fragments, and freshly deposited manure that was supplemented with 15NO3-. We observed that the 15N content of fungal hyphae grown on the slides was significantly lower in regions where the hyphae were influenced by N-rich manure than in regions influenced by N-deficient straw. This effect occurred over distances of tens to hundreds of microns. Our data illustrate that TOF-SIMS has the potential to locate N-assimilating microorganisms in soil and to quantify the 15N content of cells that have assimilated 15N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.

Carbon↗
PubMed1974

Ultrastructure of a thermotolerant basidiomycete possibly suitable for production of food protein.

The imperfect cellulolytic fungus Sporotrichum pulverulentum, which is commonly found growing in wood-chip piles, was grown in submerged culture on wheat shorts and other cereal flours. These substrates were broken down in 1 to 4 days at 30 to 40 C, and the mycelial mass was easily harvested by filtration. Scanning electron micrographs of hyphae in mycelial pellets are presented, and thin sections of conidia and hyphae were studied in a transmission electron microscope. Dolipores in septa of hyphae were observed, and cell walls are shown to be lamellar, which is characteristic of the Basidiomycetes. Actively growing hyphae are full of cytoplasm with numerous mitochondria, whereas old mycelial pellets contain highly vacuolated and almost empty cells.

Age Factors↗
PubMed1978

Scanning electron microscopy of experimental Trichophyton mentagrophytes infections in guinea pig skin.

Trichophyton mentagrophytes invasion of guinea pig skin was examined by scanning electron microscopy. Biopsies were obtained daily for 12 days from experimental infection sites. Dermatophyte invasion, examined in detail by scanning electron microscopy of cross-sectioned, prefixed skin was evidenced by: the appearance of hyphae within the stratum corneum; follicular invasion by hyphae, which remained initially within the follicle wall; emergence of the hyphae from the wall into the follicular canal; proliferation of the fungus down the follicle, with furrowing of the follicle wall and hair shaft cuticle; penetration of hyphae into the hair shaft by subcuticular and transcuticular routes; and massive peripilar hyphal proliferation with arthrosporogenesis. A three-dimensional perception of the invasion sequence of a dermatophyte in guinea pig skin was obtained by scanning electron microscopy.

Animals↗