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PubMed2006

Candida albicans Ecm33p is important for normal cell wall architecture and interactions with host cells.

Candida albicans ECM33 encodes a glycosylphosphatidylinositol-linked cell wall protein that is important for cell wall integrity. It is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis. To identify potential mechanisms through which Ecm33p contributes to virulence, we investigated the interactions of C. albicans ecm33Delta mutants with endothelial cells and the FaDu oral epithelial cell line in vitro. The growth rate of blastospores of strains containing either one or no intact copies of ECM33 was 50% slower than that of strains containing two intact copies of ECM33. However, all strains germinated at the same rate, forming similar-length hyphae on endothelial cells and oral epithelial cells. Strains containing either one or no intact copies of ECM33 had modestly reduced adherence to both types of host cells, and a markedly reduced capacity to invade and damage these cells. Saccharomyces cerevisiae expressing C. albicans ECM33 did not adhere to or invade epithelial cells, suggesting that Ecm33p by itself does not act as an adhesin or invasin. Examination of ecm33Delta mutants by transmission electron microscopy revealed that the cell wall of these strains had an abnormally electron-dense outer mannoprotein layer, which may represent a compensatory response to reduced cell wall integrity. The hyphae of these mutants also had aberrant surface localization of the adhesin Als1p. Collectively, these results suggest that Ecm33p is required for normal cell wall architecture as well as normal function and expression of cell surface proteins in C. albicans.

Candida albicans↗
PubMed2006

The Cek1 and Hog1 mitogen-activated protein kinases play complementary roles in cell wall biogenesis and chlamydospore formation in the fungal pathogen Candida albicans.

The Hog1 mitogen-activated protein (MAP) kinase mediates an adaptive response to both osmotic and oxidative stress in the fungal pathogen Candida albicans. This protein also participates in two distinct morphogenetic processes, namely the yeast-to-hypha transition (as a repressor) and chlamydospore formation (as an inducer). We show here that repression of filamentous growth occurs both under serum limitation and under other partially inducing conditions, such as low temperature, low pH, or nitrogen starvation. To understand the relationship of the HOG pathway to other MAP kinase cascades that also play a role in morphological transitions, we have constructed and characterized a set of double mutants in which we deleted both the HOG1 gene and other signaling elements (the CST20, CLA4, and HST7 kinases, the CPH1 and EFG1 transcription factors, and the CPP1 protein phosphatase). We also show that Hog1 prevents the yeast-to-hypha switch independent of all the elements analyzed and that the inability of the hog1 mutants to form chlamydospores is suppressed when additional elements of the CEK1 pathway (CST20 or HST7) are altered. Finally, we report that Hog1 represses the activation of the Cek1 MAP kinase under basal conditions and that Cek1 activation correlates with resistance to certain cell wall inhibitors (such as Congo red), demonstrating a role for this pathway in cell wall biogenesis.

Candida albicans↗
PubMed2002

Developmental regulation of the Streptomyces lividans ram genes: involvement of RamR in regulation of the ramCSAB operon.

Streptomycetes are filamentous soil bacteria that produce spores through a complex process of morphological differentiation. The ram cluster plays an important part during the development. The ram genes encode a membrane-bound kinase (RamC), a small protein (RamS), components of an ABC transporter (RamAB), and a response regulator (RamR). While the introduction of an extra copy of the ram cluster accelerates development in Streptomyces lividans, ramABR disruption mutants are unable to produce aerial hyphae and spores. The developmental regulation of ram gene transcription was analyzed. Transcription of the ram genes occurred only on solid rich media and not on minimal media. The ramR gene is transcribed from a single promoter during all growth stages, with the highest levels during aerial growth. The ramCSAB genes comprise one operon and are transcribed from one principal promoter, P1, directly upstream of ramC. Transcription of ramCSAB was already observed during vegetative growth, but was strongly upregulated upon initiation of formation of aerial hyphae and was decreased during late stages of development. A large inverted repeat located downstream of ramS terminated the majority of transcripts. The introduction of ramR on a multicopy vector in S. lividans strongly induced P1 activity, while disruption of this regulator eliminated all P1 promoter activity. This shows that ramR is a crucial activator of ramCSAB transcription. Importantly, in bldA, bldB, bldD, or bldH mutants, ramR and ramCSAB are not transcribed, while ram gene transcription was observed in the earliest whi mutant, whiG. This indicates that the transcription of the ram genes marks the transition from vegetative to aerial growth.

ATP-Binding Cassette Transporters↗
PubMed2005

The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans.

Formins are conserved eukaryotic proteins playing key roles in regulating cell polarity. We have characterized the roles of a formin CaBni1p in the polymorphic fungus Candida albicans. CaBni1p localized persistently to hyphal tips during hyphal growth but to distinct growth sites at different cell cycle stages during yeast growth. Cabni1Delta yeast cells exhibited several morphological defects, such as round and enlarged cells, widened bud necks and a random budding pattern. Although Cabni1Delta cells could still undergo yeast-hypha growth switch, the hyphae were markedly swollen. Cabni1Delta also showed defects in spindle and cytoplasmic microtubule orientation and positioning. Coincidentally, the spindle orientation protein CaKar9p in Cabni1Delta yeast cells appeared as multiple random cortical spots, in contrast to the single spot at the bud tip of many wild-type cells. Interestingly, several defects manifested in Cabni1Delta yeast cells were partially corrected during hyphal growth. We found that the second formin CaBnr1p was recruited to hyphal tips, while it localized only to the bud neck during yeast growth. This behavior of CaBnr1p may play a key role in correcting Cabni1Delta defects during hyphal growth. Cabni1Delta exhibited reduced virulence in mice. These results indicate that the formins play an important role in Candida albicans polarized growth and CaBni1p's function is required for virulence.

Actins↗
PubMedDate not supplied

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin.

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.

Carbohydrates↗
PubMedDate not supplied

Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi.

Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.

Daucus carota↗
PubMed2005

Pseudofungi in pericolic lymph nodes.

We report a case of extensive pseudofungi in the pericolic lymph nodes. A 45-year-old man presented with lower abdominal discomfort, and large solid and cystic masses were discovered in the pelvic cavity by computed tomography. Debulking of the masses (by means of a Hartmann operation) was performed under the clinical impression of a pelvic sarcoma, and histologically the masses demonstrated features of a malignant gastrointestinal stromal tumor. All pericolic lymph nodes demonstrated multiple septate hyphae-like structures with pigmentation, particularly in the subcapsular sinuses. These structures showed positive staining with periodic acid-Schiff, but Gomori methenamine silver staining was negative. The structures appeared to be composed of iron, phosphorus, and calcium by Perls iron and von Kossa stains and revealed deposition of electron-dense granules on electron microscopic examination. Energy-dispersive radiographic micro-analysis showed the structures to contain phosphorus, sulfur, potassium, calcium, and iron. Pseudofungi can be misinterpreted as true fungi with septate hyphae; therefore, a careful morphologic examination with appropriate special stains is mandatory.

Diagnosis, Differential↗
PubMed2002

Monoxenic in vitro production and colonization potential of AM fungus Glomus intraradices.

The paper reports the establishment of mycorrhizal infection of a non-mycorrhizal Ri-T-DNA transformed carrot root when co-cultured with a surface sterilized sweet potato root segment colonized by arbuscular mycorrhizal (AM) fungus G. intraradices on minimal M medium. Extensive fungal hyphal emergence from each cut end of the mycorrhizal sweet potato root piece was observed in one week old cultures. These hyphae caused infection on contacting the transformed-carrot- root segment and produced many hyphae and spores both inside and outside the zone of the root after 6 week of growth. Axenically produced fungal propagules proliferated on the surface of fresh minimal M medium when sub-cultured without any root segment. On repeated sub-culturing, these propagules did not lose their ability to grow and produced many juvenile small spore-like vesicles during the non-symbiotic phase. Although these spores were morphologically and anatomically similar to their soil borne counter parts, they were much smaller. When placed in the vicinity of a fresh hairy root on the minimal medium or a Sudan grass seedling in sand culture, the axenically produced AM fungal propagules caused root infection, but the infection characteristics were significantly different to the original culture in terms of shape (spherical vs oval) and size (20 microm vs 45 microm) of the intraradical vesicles, and absence of 'H' branches. Sudan grass seedlings inoculated with the axenically cultured fungus showed significantly (P < 0.05) higher dry weights plant'. When compared to the plants inoculated with sand cultures, the growth parameters and the percentage infection were not significantly different. However, when both sources of inocula were used together, a synergistic effect on plant growth as well as root infection was observed.

Coculture Techniques↗
PubMed2002

Oils activity in the control of rose powdery mildew.

The efficacy and eventual phytotoxicity of oils from rape, sunflower seed (vegetable oil) and paraffin (Atpolan 80 EC) in the control of Sphaerotheca pannosa var. rosae were evaluated on roses grown in greenhouse or plastic tunnel. Oils were applied curativelly as plant spray 4-times at 7-day-intervals in concentrations from 0.25 to 4%. All used oils, gave as good effect as triforine (standard) or even better. On shrubs protected with oils at concentrations 0.25% and 0.5%, disease symptoms were noted only sporadically. Rose shrubs sprayed with higher concentration of oils were almost completely protected. Efficacy of tested oils increased with their concentrations. Paraffin oil was better in the control of S. pannosa var. rosae than vegetable oils. After 2 oil applications at conc. 2% or higher, first phytotoxicity symptoms on leaves were noted, particularly during warm and sunny days. Observations of treated leaves, 24 h after spraying under microscope, showed that all tested oils caused hyphae and spores degeneration or their visible deformation suggesting partial loss of turgidity. Depends on oil concentrations used, sporadically regular and unchanged shape hyphae and spores were visible.

Ascomycota↗
PubMedDate not supplied

[Spontaneous sector formation in soil streptomycetes colonies].

A model of the formation of sectors during the growth and zone formation of a colony of the soil radiant fungus streptomycetes was developed. It was shown that the basic parameters determining the shape of the sector border are the correlations between the growth rates of the sector of mycelium hyphae and the remainder of the colony and between the frequency and the numbers of branching order of these hyphae. Based on the long-standing observations, a polyseasonal dependence of spontaneous sector formation in streptomycetes zone-forming colonies was demonstrated.

Hyphae↗
PubMed2005

[Field distribution pattern and metabolic activity of AM fungi and their effects on Ipomoea batatas growth].

The study showed that after eight weeks of inoculating sweet potato seedlings with AM fungus Glomus intraradices BEG141 under field condition, the shoot dry weight, individuals of tuber and their fresh weight, as well as the P uptake by the plant increased significantly. Compared with the control, the root colonization rate, extraradical hyphal density and intraradical alkaline phosphatase (ALP) activity were greatly increased. After inoculation with BEG141, the total and succinate dehydrogenase (SDH)- active hyphae paralleling with ridge were much more than those in underside direction, while there was no significant difference in different directions in the control. The increase of intra- and extraradical active hyphae density and the change of AM fungus distribution pattern after inoculation with mycorrhizal fungi may be the main reasons for the growth improvement of sweet potato.

Alkaline Phosphatase↗
PubMed2006

[A new bioassay model of antibiotic susceptibility by corneofungimetry].

OBJECTIVE: To study the value of a new bioassay model of antibiotic susceptibility by corneofungimetry which is more similar to human internal environment. METHODS: Twenty-one healthy adult volunteers, 12 males and 9 females, aged 32 (22 - 41), were randomly divided into two groups to receive itraconazole 200 mg Bid or 200 mg qd or bid for one week. Stratum corneum strippings were taken from the forearm skin once before administration of itraconazole and 1, 4, 7, 10, 14, 21, 28, and 35 days after administration of itraconazole. Spores of selected fungi (Trichophyton rubrum, T. mentagrophytes, Microsporum canis, Candida albicans, C. glabrata and C. tropicalis) were deposited and cultured on the strippings. PAS stain was used after one-week culture to observe the growth of the fungi by computerized-assisted image analysis to measure the area covered by the yeasts and the dermatophyte. RESULTS: All fungi grew on all the stratum corneum strippings with different numbers of hyphae. The areas covered by hyphae and spores decreased gradually after administration of itraconazole, became the smaller on the seventh day (P = 0.01 or P < 0.01), and began to increased since the 10 th day, and became almost the same sizes as those before the administration of itraconazole on the 35 th day. CONCLUSION: Similar to the human internal environment, corneofungimetry is a useful new model of bioassay for antibiotic susceptibility.

Adult↗
PubMed2006

Role of CaBud6p in the polarized growth of Candida albicans.

Bud6p is a component of a polarisome that controls cell polarity in Saccharomyces cerevisiae. In this study, we investigated the role of the Candida albicans Bud6 protein (CaBud6p) in cell polarity and hyphal development. CaBud6p, which consists of 703 amino acids, had 37% amino-acid sequence identity with the Bud6 protein of S. cerevisiae. The homozygous knock-out of CaBUD6 resulted in several abnormal phenotypes, such as a round and enlarged cells, widened bud necks, and a random budding pattern. In hypha-inducing media, the mutant cells had markedly swollen tips and a reduced ability to switch from yeast to hypha. In addition, a yeast two-hybrid analysis showed a physical interaction between CaBud6p and CaAct1p, which suggests that CaBud6p may be involved in actin cable organization, like Bud6p in S. cerevisiae. Taken together, these results indicate that CaBud6 plays an important role in the polarized growth of C. albicans.

Actins↗
PubMed1997

A new classification and diagnostic criteria for invasive fungal sinusitis.

OBJECTIVE: To develop criteria for the diagnosis of invasive fungal sinusitis. DESIGN: Review of the literature on invasive fungal sinusitis in the context of a population of 30 patients with fungal sinusitis and 24 patients with chronic bacterial sinusitis. SETTING: Tertiary care medical center. RESULTS: Our review revealed no consensus in the literature on the classification of the syndromes of invasive fungal sinusitis and no criteria for their diagnosis. Moreover, the existing syndromes of invasive fungal sinusitis lacked specificity and one of the more commonly cited syndromes, primary aspergillosis of the paranasal sinuses, is a granulomatous disease that occurs rarely outside Africa. Two of our 30 patients with fungal sinusitis had a previously unrecognized form of invasive disease. Both were middle-aged adults with well-controlled type 2 diabetes mellitus, apical orbital syndrome, and a similar course: proptosis resulting from fungal expansion out of an ethmoid sinus, a protracted illness of 6 months or longer, visual changes, late neurological symptoms reflecting cavernous sinus invasion, and death. The syndrome in these 2 patients is distinct from the syndrome of fulminant invasive fungal sinusitis, (eg, mucormycosis) with nasal eschar, intracerebral fungal dissemination by vascular invasion, and death in days, and the granulomatous form. CONCLUSIONS: We conclude that there are 3 forms of invasive fungal sinusitis and propose that they be termed (1) granulomatous, (2) acute fulminant, and (3) chronic invasive. The latter category reflects the syndrome seen in our 2 patients. Furthermore, the following 2 diagnostic criteria for invasive fungal sinusitis are proposed: (1) sinusitis confirmed by radiological imaging and (2) histopathological evidence of hyphal forms within sinus mucosa, submucosa, blood vessels, or bone. The specificity of hyphae within sinus mucosa for tissue invasion was supported by the absence of stainable hyphae in the mucosa of patients with chronic bacterial sinusitis or in the mucosa of our described patients with allergic fungal sinusitis and mycetoma.

Adult↗
PubMed1999

Eleven novel sep genes of Schizosaccharomyces pombe required for efficient cell separation and sexual differentiation.

Genetic analysis of 20 sterile mutants prone to form hyphae revealed 11 novel ste genes (sep6 to sep16) of Schizosaccharomyces pombe. None of the mutants was completely mycelial. Most mutants formed branching hyphae and showed normal septation. Aberrant septal structures and actin distribution were seen only at 36 degrees C. sep9-307, sep14-576 and sep15-598 showed genetic interactions with sep1-1, a mutation in a forkhead transcription factor homologue. Additional genetic interactions were detected between sep6-194, sep15-598 and cdc16-116, a mutant allele of an anaphase modulator of p34cdc2. sep9-307 and sep15-598 caused dikaryosis in wee1- background. In mating and sporulation tests, sep6-, sep7-, sep9-, sep10-, sep11- and sep15- proved to be defective in conjugation only, whereas sep8-, sep13- and sep16- were also defective in meiosis-sporulation. sep12- and sep14- were only partially sterile. All mutants could produce M-factor but sep8-, sep11-, sep15- and sep16- were defective in P-factor production. The mutations in sep8, sep11 and sep16 suppressed the pat1-114-driven meiosis. All mutants were sensitive to the presence of higher concentrations of chloride in the medium and to short heat shocks. The diversity of the mutant phenotypes and the pleiotropic effects of the mutations suggest that these sep genes might act in, or interact with, a multiple overlapping network of regulatory modules.

Actins↗
PubMed2000

Two-photon autofluorescence microscopy and spectroscopy of Antarctic fungus: new approach for studying effects of UV-B irradiation.

We combined two-photon fluorescence microscopy and spectroscopy to provide functional images of UV-B (280-315 nm) induced stress on an Antarctic fungus. Two-photon excitation microscopy was used to characterize the distribution of autofluorescence inside the spore and the hyphae of the fungus. The imaging analysis clearly shows that the autofluorescence response of spores is higher than that of hyphae. The imaging analysis at different depths shows that, strikingly enough, the spore autofluorescence originates from the cell wall and membrane fluorophores. The spectroscopic results show moreover that the fluorescence spectra of spores are redshifted upon UV-B irradiation. Tentative identification of the chromophores involved in the autofluorescence response and their biological relevance are also discussed on the basis of a previous steady-state fluorescence spectroscopic study performed on both whole spore suspension and organic-soluble extracts.

Image Processing, Computer-Assisted↗
PubMed2000

Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale.

It is well known that high-viscosity fermentation broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some processes, this has been shown to damage mycelia, affect morphology, and decrease product expression. However, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pilot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m(3) fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation function (EDCF) and was found to have average values of 29.3 +/- 1.0 and 22.0 +/- 0.3 kW m(-3) s(-1) at high and low power levels, respectively. In all batches, biomass concentration profiles were similar and specific growth rate was < 0.03 h(-1). Morphological data show hyphal fragmentation occurred by both shaving-off of external clump hyphae and breakage of free hyphae. The fragmentation rate constant (k(frag)), determined using a first-order model, was 5.90 and 5.80 h(-1) for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal elements. In all batches, fragmentation was found to dominate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process.

Aspergillus oryzae↗
PubMed2002

Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence-based digital image analysis.

Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus. After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen. The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera. Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 microg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells. Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA-stained area) by image analysis. At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA-stained area with its growth rate, a functional indicator of viability. The linear correlation (r = 0.979) was: growth rate (g/L x h) = 2.25 x 10(-3) (% of FDA-stained area). This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10-L bioreactor. Estimated viable biomass during fermentation was strongly influenced by the process conditions. The use of FDA, with computer-aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T. harzianum.

Acridine Orange↗