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PubMed2004

Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365.

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.

Chemotactic Factors↗
PubMed2004

The SC15 protein of Schizophyllum commune mediates formation of aerial hyphae and attachment in the absence of the SC3 hydrophobin.

Disruption of the SC3 gene in the basidiomycete Schizophyllum commune affected not only formation of aerial hyphae but also attachment to hydrophobic surfaces. However, these processes were not completely abolished, indicating involvement of other molecules. We here show that the SC15 protein mediates formation of aerial hyphae and attachment in the absence of SC3. SC15 is a secreted protein of 191 aa with a hydrophilic N-terminal half and a highly hydrophobic C-terminal half. It is not a hydrophobin as it lacks the eight conserved cysteine residues found in these proteins. Besides being secreted into the medium, SC15 was localized in the cell wall and the mucilage that binds aerial hyphae together. In a strain in which the SC15 gene was deleted (DeltaSC15) formation of aerial hyphae and attachment were not affected. However, these processes were almost completely abolished when the SC15 gene was deleted in the DeltaSC3 background. The absence of aerial hyphae in the DeltaSC3DeltaSC15 strain can be explained by the inability of the strain to lower the water surface tension and to make aerial hyphae hydrophobic.

Amino Acid Sequence↗
PubMed2005

Differential cytokine production and Toll-like receptor signaling pathways by Candida albicans blastoconidia and hyphae.

Toll-like receptors (TLR) are crucial for an efficient antifungal defense. We investigated the differential recognition of blastoconidia and hyphae of Candida albicans by TLRs. In contrast to Candida blastoconidia, which stimulated large amounts of gamma interferon (IFN-gamma), the tissue-invasive Candida hyphae did not stimulate any IFN-gamma by human peripheral blood mononuclear cells (PBMC) or murine splenic lymphocytes. After stimulation with blastoconidia, the production of IFN-gamma was TLR4 dependent, as shown by the significantly decreased IFN-gamma production in anti-TLR4-treated PBMC and in splenic lymphocytes from TLR4-defective ScCr mice. In addition, peritoneal macrophages from ScCr mice produced less tumor necrosis factor alpha (TNF-alpha) than macrophages of control mice did when stimulated with Candida blastoconidia, but not with hyphae, indicating that TLR4-mediated signals are lost during hyphal germination. In contrast, macrophages from TLR2 knockout mice had a decreased production of TNF-alpha in response to both Candida blastoconidia and hyphae. Candida hyphae stimulated production of interleukin-10 through TLR2-dependent mechanisms. In conclusion, TLR4 mediates proinflammatory cytokine induction after Candida stimulation, whereas Candida recognition by TLR2 leads mainly to anti-inflammatory cytokine release. TLR4-mediated proinflammatory signals are lost during germination of Candida blastoconidia into hyphae. Phenotypic switching during germination may be an important escape mechanism of C. albicans, resulting in counteracting host defense.

Animals↗
PubMed2005

Candida albicans hyphae have a Spitzenkörper that is distinct from the polarisome found in yeast and pseudohyphae.

Fungi grow with a variety of morphologies: oval yeast cells, chains of elongated cells called pseudohyphae and long, narrow, tube-like filaments called hyphae. In filamentous fungi, hyphal growth is strongly polarised to the tip and is mediated by the Spitzenkörper, which acts as a supply centre to concentrate the delivery of secretory vesicles to the tip. In the budding yeast Saccharomyces cerevisiae, polarised growth is mediated by the polarisome, a surface cap of proteins that nucleates the formation of actin cables delivering secretory vesicles to the growing tip. The human fungal pathogen, Candida albicans, can grow in all three morphological forms. Here we show the presence of a Spitzenkörper at the tip of C. albicans hyphae as a ball-like localisation of secretory vesicles, together with the formin Bni1 and Mlc1, an ortholog of an S. cerevisiae myosin regulatory light chain. In contrast, in C. albicans yeast cells, pseudohyphae and hyphae Spa2 and Bud6, orthologs of S. cerevisiae polarisome components, as well as the master morphology regulator Cdc42, localise predominantly, but not exclusively, to a surface cap resembling the polarisome of S. cerevisiae yeast cells. A small amount of Cdc42 also localises to the Spitzenkörper. Furthermore, we show differences in the genetic and cytoskeletal requirements, and cell cycle dynamics of polarity determinants in yeast, pseudohyphae and hyphae. These results, together with the cytological differences between the cell types, suggest that the Spitzenkörper and polarisome are distinct structures, that the polarisome and Spitzenkörper coexist in hyphae, and that polarised growth in hyphae is driven by a fundamentally different mechanism to that in yeast and pseudohyphae.

Candida albicans↗
PubMed2001

Strength of mid-logarithmic and stationary phase Saccharopolyspora erythraea hyphae during a batch fermentation in defined nitrate-limited medium.

A method for measuring mechanical properties of Saccharopolyspora erythraea is reported with data from a batch fermentation. Briefly, hyphae were glued to the end of a tungsten filament mounted horizontally on a sensitive force transducer. Free ends of hyphae were trapped against a flat surface by a second probe. The force transducer and tungsten filament were then moved at a fixed rate, the hypha were strained, and the force resisting motion recorded. From these data the maximum force resisting motion is taken as the force at which breakage occurs. Hyphae from the mid-logarithmic phase of a simple batch fermentation on defined medium were found to have a breaking force of 890 +/- 160 nN (95% confidence), while stationary phase hyphae were weaker at 580 +/- 150 nN. Video recordings of the experiments allowed an approximation of breaking strain, which did not differ significantly between samples at 0.18 +/- 0.03. Electron microscopy was used to measure cell wall thickness, cell diameter, and hence cell wall cross-sectional area. The ultimate tensile strength was estimated to be 24 +/- 3 MPa with no difference between the two samples, the lower breaking force of the stationary phase hyphae being attributed to a thinner cell wall. Assuming a linear relationship between stress and strain, the elastic modulus was estimated to be 140 +/- 30 MPa. These values are comparable with other structural biological materials such as yeast cell walls and collagen.

Anti-Bacterial Agents↗
PubMedDate not supplied

What determines growth direction in fungal hyphae?

We used high-resolution video microscopy and image analysis to map the trajectory of the Spitzenkörper in growing hyphae of Neurospora crassa and to correlate it with growth directionality. The Spitzenkörper followed a tortuous trajectory produced by a dominant forward motion accompanied by frequent, transverse oscillations. In hyphae with a fixed growth direction, the regression line of the Spitzenkörper trajectory corresponded to the longitudinal axis of the hypha. A permanent change in growth direction, i.e., the establishment of a new growth axis, was correlated with a sustained shift in Spitzenkörper trajectory away from the existing cell axis. In meandering hyphae, changes in growth directionality occurred somewhat erratically but there was a strong compensatory tendency reversing directional shifts and maintaining an overall fixed direction of growth. Although external factors greatly affect hyphal growth direction (tropisms), they are probably not the primary determinants of growth directionality. Inhibitors of microtubules, but not of actin microfilaments, caused hyphae to lose their growth directionality-providing support for the idea that Spitzenkörper trajectory is determined internally by a growing scaffolding of cytoplasmic microtubules. The meandering morphology of N. crassa hyphae was duplicated by computer simulation in support of the idea that hyphal morphogenesis is controlled by the position of the Spitzenkörper functioning as a vesicle supply center.

Computer Simulation↗
PubMed2005

Carbon source induced yeast-to-hypha transition in Candida albicans is dependent on the presence of amino acids and on the G-protein-coupled receptor Gpr1.

Yeast-to-hypha transition in Candida albicans can be induced by a wide variety of factors, including specific nutrients. We have started to investigate the mechanism by which some of these nutrients may be sensed. The G-protein-coupled receptor Gpr1 is required for yeast-to-hypha transition on various solid hypha-inducing media. Recently we have shown induction of Gpr1 internalization by specific amino acids, e.g. methionine. This suggests a possible role for methionine as a ligand of CaGpr1. Here we show that there is a big variation in methionine-induced hypha formation depending on the type of carbon source present in the medium. In addition high glucose concentrations repress hypha formation whereas a concentration of 0.1%, which mimics the glucose concentration present in the bloodstream, results in maximal hypha formation. Hence, it remains unclear whether Gpr1 senses sugars, as in Saccharomyces cerevisiae, or specific amino acids like methionine.

Amino Acids↗
PubMed1988

Induction of signal transduction in human neutrophils by Candida albicans hyphae: the role of pertussis toxin-sensitive guanosine triphosphate-binding proteins.

Nonopsonized Candida hyphae elicit from human neutrophils a transient rise in cytosolic calcium concentrations and an oxidative burst without a detectable change in membrane potential. To determine if the signal-transduction pathway used by these organisms is mediated by guanine nucleotide-binding proteins (GNPs), we examined the functional responsiveness of neutrophils pretreated with pertussis toxin (PT). In response to serum-opsonized hyphae or zymosan, the rise in cytosolic calcium, membrane depolarization, and the respiratory burst were only partially abrogated. The transient rise in calcium induced by unopsonized hyphae was, however, completely eliminated in PT-treated neutrophils. Despite total abrogation of the calcium response, PT-treated cells could still mount a respiratory burst in response to these nonopsonized hyphae. Thus, neutrophil signaling by both serum-opsonized particles and nonopsonized hyphae is only partially mediated by PT-sensitive GNPs. Furthermore, the ability of unopsonized hyphae to elicit a respiratory burst without a calcium response suggests these events are separable and confirms the versatility of these organisms as probes for investigating neutrophil activation.

Calcium↗
PubMed1990

Patterns of guanine nucleotide exchange reflecting disparate neutrophil activation pathways by opsonized and unopsonized Candida albicans hyphae.

Through guanosine triphosphate (GTP) regulatory proteins are crucial components in signal transduction by most soluble and opsonized particulate stimuli, previous data suggest that neutrophil (PMNL) activation by unopsonized hyphae differs. Most of the PMNL superoxide response evoked by unopsonized hyphae was independent of both Ca++ ions and pertussis toxin-sensitive guanine nucleotide regulatory proteins. To determine whether related regulatory proteins were involved in PMNL activation by unopsonized hyphae, separated PMNL plasma membranes were incubated with GTP and a poorly hydrolyzed, radiolabeled GTP analogue, 5'-guanylylimido-diphosphate, then stimulated. Particulate Candida albicans hyphae and soluble chemotactic peptide induced comparable guanine nucleotide release. In contrast, while unopsonized hyphae caused release, it was considerably delayed, though opsonization discernibly affected neither PMNL attachment nor spreading over hyphal surfaces. This paralleled earlier observations of other delayed responses by intact PMNL to unopsonized hyphae: phospholipase C activation, the rise in cytosolic free Ca++ ions, and actin polymerization.

Candida albicans↗
PubMed1991

In vivo interferon-gamma therapy augments the in vitro ability of chronic granulomatous disease neutrophils to damage Aspergillus hyphae.

During the recently completed double-blind, placebo-controlled, randomized trial of recombinant interferon-gamma (rIFN-gamma) therapy in chronic granulomatous disease (CGD), a metabolic assay of neutrophil damage to Aspergillus fumigatus hyphae was used to monitor neutrophil function before and during therapy. In this assay, 5 x 10(4) conidia that had germinated into hyphae were exposed to 5 x 10(5), 15 x 10(5), or 50 x 10(5) CGD neutrophils. By analysis of variance, neutrophils from patients on rIFN-gamma were found to produce significantly more damage to hyphae than those from the placebo group (P less than .01). In subgroup analysis, this effect was best seen in the hyphae exposed to 50 x 10(5) CGD neutrophils, where neutrophils from patients receiving rIFN-gamma produced significantly more damage to the hyphae than those from the placebo group (P less than .05). In vivo rIFN-gamma therapy improves the ability of CGD neutrophils to damage Aspergillus fumigatus hyphae in an in vitro assay.

Analysis of Variance↗
PubMed1986

Differences in actin localization during bud and hypha formation in the yeast Candida albicans.

Stationary phase cells of Candida albicans can form either a bud or a hypha, depending upon the pH of the medium into which they are released. At low pH, cells form an ellipsoidal bud and at high pH, cells form an elongated hypha. By staining cells with rhodamine-conjugated phalloidin, we have compared the dynamics of actin localization during the formation of buds and hyphae. Before evagination, actin granules were distributed throughout the cytoplasmic cortex in both budding and hypha-forming cells. Just before evagination, actin granules clustered at the site of evagination, then filled the early evagination in both budding and hypha-forming cells. With continued bud growth, the actin granules then redistributed throughout the cytoplasmic cortex. In marked contrast, with continued hyphal growth, the majority of actin granules clustered at the hyphal apex. This distinct difference in actin granule localization may be related to the distinct differences in the expansion zones of the cell wall recently demonstrated between growing buds and hyphae. The spatial and temporal dynamics of the large neck actin granules and of actin fibres are also described.

Actins↗
PubMed1997

Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor.

In the filamentous bacterium Streptomyces coelicolor, the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 microm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 microm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants (whiG, whiH and whiB), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.

Antibodies, Bacterial↗
PubMed1987

Scanning Electron Microscope Studies of Interactions between Agaricus bisporus (Lang) Sing Hyphae and Bacteria in Casing Soil.

Relationships between the hyphae of Agaricus bisporus (Lang) Sing and bacteria from the mushroom bed casing layer were examined with a scanning electron microscope. Hyphae growing in the casing layer differed morphologically from compost-grown hyphae. Whereas the compost contained thin single hyphae surrounded by calcium oxalate crystals, the casing layer contained mainly wide hyphae or mycelial strands without crystals. The bacterial population in the hyphal environment consisted of several types, some attached to the hyphae with filamentlike structures. This attachment may be important in stimulation of pinhead initiation.

Journal Article↗
PubMed1995

Quantification of fungal hyphae in leaves of deciduous trees by automated image analysis.

An optical method to quantify the fungal hyphae within decomposing leaves of deciduous trees was developed. The plant matrix was partially destroyed under hydrolytic conditions, and fungal hyphae and cellulose residues within the leaves were stained with Calcofluor M2R. Cellulose residues were subsequently depolymerized by cellulase, and fungal hyphae were separated from the remaining plant matrix with a pressurized air-water mixture. An image analysis program to quantify the fungal hyphae was written. The program included the recognition of fungal hyphae, the elimination of stomata from the images, and the measuring of lengths of fungal hyphae. The optical method was verified by a chemical method relying on glucosamine as an indicator of fungal biomass. The fungal biomass in leaves of Fagus silvatica and Quercus petraea at early states of decomposition was 0.2 to 0.4% of the leaf weight. The biomass reached a maximum within 2 to 4 weeks (optical method, 0.5 to 0.7%; chemical method, 1 to 1.4% of the initial leaf weight) and decreased thereafter.

Journal Article↗
PubMed2001

Growth of chitinolytic dune soil beta-subclass Proteobacteria in response to invading fungal hyphae.

It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse living fungal hyphae, thereby releasing potential growth substrate. However, the conditions used in such assays (high bacterial density, rich media, fragmented hyphae) make it difficult to determine whether mycolytic activity is actually of importance for the growth and survival of chitinolytic bacteria in soils. An unidentified group of beta-subclass Proteobacteria (CbetaPs) was most dominant among the culturable nonfilamentous chitinolytic bacteria isolated from Dutch sand dune soils. Here we demonstrate that the CbetaPs grew at the expense of extending fungal mycelium of three dune soil fungi (Chaetomium globosum, Fusarium culmorum, and Mucor hiemalis) under nutrient-limiting, soil-like conditions. Aggregates of CbetaPs were also often found attached to fungal hyphae. The growth of a control group of dominant nonchitinolytic dune soil bacteria (beta- and gamma-subclass Proteobacteria) was not stimulated in the mycelial zone, indicating that growth-supporting materials were not independently released in appreciable amounts by the extending hyphae. Therefore, mycolytic activities of CbetaPs have apparently been involved in allowing them to grow after exposure to living hyphae. The chitinase inhibitor allosamidin did not, in the case of Mucor, or only partially, in the cases of Chaetomium and Fusarium, repress mycolytic growth of the CbetaPs, indicating that chitinase activity alone could not explain the extent of bacterial proliferation. Chitinolytic Stenotrophomonas-like and Cytophaga-like bacteria, isolated from the same dune soils, were only slightly stimulated by exposure to fungal hyphae.

Acetylglucosamine↗
PubMed1995

Production of polyclonal and monoclonal antibodies against hyphae from arbuscular mycorrhizal fungi.

Polyclonal and monoclonal antibodies were produced against hyphae of the arbuscular mycorrhizal fungus Glomus monosporum. The polyclonal antibodies (pAbs) were raised in a rabbit by immunizing with hyphae. They were tested for their specificity by a dot-immunoblot assay (DIBA). After the third immunization, a distinct difference in the signal strength was observed between the antisera and the preimmune serum. The pAbs showed cross-reactions to a number of fungal species, both mycorrhizal and other. For the production of monoclonal antibodies (mAbs), mice were immunized intraperitoneally with hyphae. The resulting hybridoma cell culture supernatants were tested by an indirect immunolabeling procedure. For this purpose the hyphae were immobilized on silane-coated microscopic slides. The mAb 8A7 reacted with hyphae from all Glomus isolates tested so far. Cross-reactivities were not observed with hyphae from fungi of the family Acaulosporaceae, phytopathogenic fungi tested so far, or from spores from Glomus species.

Animals↗
PubMedDate not supplied

[Fine structure of the vegetative and spore-forming hyphae of Micropolyspora fascifera].

Changes in the fine structure of the hyphae were studied in the course of spore formation by Micropolyspora fascifera. Sporulating hyphae differ from vegetative hyphae by a less dense cytoplasm, a large zone of the nucleoid with distinctly fibrillar structure, and a thin layerless cell wall. Spore formation is accompanied with autolysis of the vegetative hyphae, which consists in vacuolization of the hyphae and appearance of a large number of tubular membrane structures within them. Spores are formed, like in Actinomyces spp., by simultaneous division of the hyphae with septa; the structure of sporulating septa is similar to that of the fragmenting mycelium in Nocardia; the structure of mature spores is similar to that in some Actinomyces spp. Therefore, M. fascifera differs from other Micropolyspora spp. not only by the chemical composition of the cells (the presence of nocardiomicolic acids) but also by their structure. Taxonomic position of the species is discussed.

Micromonosporaceae↗
PubMed1997

Analysis of electrical phenomena accompanying the growth of Neurospora crassa hyphae: theory and experiment.

To describe electrical phenomena observed in growth of Neurospora crassa hyphae, a theoretical model was developed considering the hypha as a one-dimensional electric cable with non-uniform longitudinal distribution of current sources reflecting the activity of proton pumps. A profile of the density of the pump current along the hypha is proposed, at which the results of simulation quantitatively coincide with the results of physiological experiments. The model values of energy coupling in the growth zones were estimated. The experimental dependence of the elongation rate of regenerating apical hypha fragments on their lengths was determined. Based on the comparison of these experimental results with the results of analysis of the model, the contribution of the axial metabolite transport, from the distal parts of the hypha to the apical part, to the dynamics of the apical cell growth was estimated. The possibility of evaluating the intensity of high-molecular-weight syntheses and/or accumulation of substances in granules was demonstrated. The growth rate of the regenerating hypha fragments was shown to correlate with the electric current flowing into the apical fragment 0.2-mm in length.

Cell Membrane↗