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Results for “Hyphae”

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PubMed2000

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger.

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.

Actins↗
PubMed1999

Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa.

Calcium and proton ion fluxes were mapped at the growing apices of two hyphal organisms, the oomycete Saprolegnia ferax and the ascomycete Neurospora crassa and pseudohyphal Saccharomyces cerevisiae using self-referencing ion-selective probes. S. ferax exhibited well-defined transport zones absent in N. crassa. Ca2+ fluxes were located within 8 microm of the growing hyphal tip; the net Ca2+ flux was either inward (75% of all experiments) or outward. The inward component of the net flux was inhibited by Gd3+, known to inhibit Ca2+ permeable stretch-activated channels. Because the Ca2+ flux is located at the region of maximal hyphal expansion, exocytosis may contribute to Ca2+ efflux, in addition to the stretch-activated channel mediated influx. Maximal inward H+ flux was observed 10-30 microm behind the hyphal tip where peak mitochondria densities taper off at the onset of a vacuolation zone, presumably due to highly localized H+ cotransporter activity. By contrast, N. crassa exhibited no net Ca2+ flux and a consistently inward H+ flux (93% of all experiments) that was homogeneously distributed up to 60 microm behind the hyphal apex. Both hyphal organisms have similar tip morphology and growth rates, and are reported to have tip-high cytosolic Ca2+ gradients associated with growth. Only S. ferax exhibited tip-localized Ca2+ fluxes and a well defined H+ influx zone just behind the tip. Differences in ecological habitats and cytology--S. ferax is an aquatic organism that grows as a migrating plug of cytoplasm while N. crassa is normally terrestrial with a cytoplasm-rich mycelium and highly active cytoplasmic streaming behind the growing margin--may account for the differences in the 'architecture' of ion transport occurring during the process of tip growth. Net Ca2+ efflux and H+ influx of growing S. cerevisiae pseudohyphae were also measured but localization was not possible due to small cell size.

Calcium↗
PubMed2003

Effect of increasing inoculum sizes of Aspergillus hyphae on MICs and MFCs of antifungal agents by broth microdilution method.

In order to investigate the influence of different hyphal inoculum sizes on minimal inhibition concentrations (MICs) and minimum fungicidal concentrations (MFCs) of amphotericin B (AMB), voriconazole and itraconazole, five isolates each of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus were studied using a broth microdilution method. Three inoculum sizes were used: 1 x 10(3)-5 x 10(3), 1 x 10(4)-5 x 10(4) and 1 x 10(5)-5 x 10(5) cfu/ml. MICs and MFCs were read at 24 and 48 h at 35 degrees C. For all species tested, AMB MICs and MFCs were minimally affected by inoculum size on. However inoculum size significantly affected MICs and MFCs of voriconazole and itraconazole; there was an increase of up to 6-fold in MICs and MFCs for the various aspergilli when the inoculum increased from 10(3) to 10(5) cfu/ml (P<0.05). Thus azoles showed significant inoculum effects, while AMB showed comparatively minimum inoculum effects against Aspergillus spp.

Amphotericin B↗
PubMed1998

Motoring along the hyphae: molecular motors and the fungal cytoskeleton.

This review discusses molecular motors that use the microfilament and microtubule cytoskeletal systems in filamentous fungi. There has been an explosion in our knowledge of kinesins over the past year, because of the integration of genetic and biochemical data. The recognition of possible interactions between septation genes and cytokinesis has also advanced our understanding of microfilament-based cytoskeletal systems. We review recent findings on microfilament motors, including conventional and unconventional myosins, and the microtubule motors of the kinesin family and cytoplasmic dynein. The roles that these molecules play in hyphal morphogenesis and organelle transport provide an insight into cytoskeletal-based transport systems.

Cytoskeleton↗
PubMed1984

An investigation of the role of true hypha production in the pathogenesis of experimental oral candidosis.

From 427 cases of human oral candidosis 2135 yeast clones were screened for the presence of germ tube-negative C. albicans and variants that formed only pseudohyphae in serum; one strain of each was found. The pathogenic potential of the serum-pseudohyphal and germ tube-negative C. albicans variants was investigated in the oral cavity of the rat; both variants failed to induce palatal candidosis, in contrast to a germ tube-positive C. albicans control strain. The pathogenic potential of C. albicans strains appears to be dependent on the formation of true germ tubes.

Animals↗
PubMed1989

Immunofluorescence staining of hyphae in the histopathological diagnosis of mycoses in cattle.

An immunofluorescence staining system was developed for the identification of mycelial fungi causing systemic mycoses in cattle. Antisera to Absidia corymbifera, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger were raised in rabbits. Tissue sections of experimentally infected mice were used to determine the specificity of immune staining with antisera that had been absorbed with heterologous antigens. When 46 lesions from 22 cattle with systemic mycosis were studied, the infecting agent could be classified as a zygomycete, possibly A. corymbifera, in 18 lesions (15 cattle), A. fumigatus in 10 lesions (7 cattle) and both a zygomycete and A. fumigatus in a single lesion from one animal. Diagnosis was established by light microscopy in 13 (72%) of the 18 lesions classified as zygomycosis. Conversely, a dual infection with both a zygomycete and an Aspergillus species was suspected on the basis of morphology in nine of the 10 lesions reacting exclusively and uniformly with anti-A. fumigatus antiserum. A. fumigatus may be a more frequent aetiological agent of systemic mycosis in cattle than hitherto recognized on the basis of light microscopy and mycological culture.

Animals↗
PubMedDate not supplied

Combined effect of amphotericin B and flucytosine on hyphal growth of Candida albicans estimated at a single hypha level.

The in vitro efficacy of amphotericin B and flucytosine, separately and in combination, against the hyphal growth of Candida albicans was evaluated in situ using an automatic analysing system. A colony of C. albicans was in contact with a glucose-salt medium supplemented with biotin plus calf serum (GS medium) and GS medium containing the antifungal agent, in sequence. Minimum inhibitory concentrations at single hyphal level (S-MIC) were determined based on the response that was measured. Amphotericin B S-MICs ranged from 0.8 to 0.4 microgram ml-1, and S-MIC values > 102.4 micrograms ml-1 were obtained with flucytosine when the two agents were used independently. When the two agents were used in combination, however, a synergistic interaction between the two agents at concentrations below their individual S-MICs was observed.

Amphotericin B↗
PubMed1991

Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro.

Multiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK-like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E:T) ratio- and time-dependent. Maximal levels of anti-C. albicans activity (approximately 60%) were observed after 4 h and at E:T greater than or equal to 300:1. Similar patterns of anti-C. albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA-LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA-LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA-LAK cells (E:T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA-LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.

Animals↗
PubMed1997

WO-2, a stable aneuploid derivative of Candida albicans strain WO-1, can switch from white to opaque and form hyphae.

Candida albicans strain WO-2 was isolated as a spontaneous derivative of the white-opaque switching strain WO-1. The electrophoretic karyotype of WO-2 lacks two bands which are found in the parent. These bands correspond to one homologue of chromosome 7 and to a translocation product containing parts of chromosomes 6 and 5. Probing a blot of the karyotype demonstrated that the genetic material in these bands had been lost, yielding an aneuploid strain. UV-irradiation experiments showed that auxotrophs due to mutation in genes located in this region predominated, supporting the conclusion that WO-2 is partially haploid. WO-2 contained about 10% of its genome in the haploid state, and it grew with a doubling time of about twice that of its parent. However, it was able to undergo both the yeast-to-hyphal transition and the white-opaque transition. Hence, these processes do not require perfect diploidy.

Aneuploidy↗