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PubMed1978

Direct observation of fungal aggregates in sand dune soil.

The mycorrhizal fungus Glomerus in association with bean hosts, Phaseolus vulgaris L., growing in pot cultures and grass hosts, Calamovilfa longiflora (Hook). Scribn and Andropogon sp. growing on Lake Huron sand dunes produced extensive external mycelium. This mycelium was the dominant factor in the aggregation of soil particles. Light and scanning electron microscope studies indicated that the sand grains were attached to the hyphae. An amorphous deposit was often present at the interfaces of sand grains and hyphae. It appeared to act as an adhesive. Staining procedures indicated that this material contained polysaccharide. Other microogranisms were observed in association with the Glomus hyphae and the amorphous deposits.

Bacteria↗

PubMed1981

Inhibition of Fusarium moniliforme var. subglutinans, the causal agent of pine pitch canker, by the soil bacterium Arthrobacter sp.

A species of Arthrobacter was recovered during culture of the causal organism of pitch canker of southern pines. Fusarium moniliforme var. subglutinans (FMS). Arthrobacter is a relatively common soil bacterium and is lytic to several fungal pathogens in the soil. Soil samples from two seed orchards with pitch canker and one from a healthy pine plantation all yielded Arthrobacter. These isolates were evaluated for their ability to inhibit the growth of FMS isolates from pitch canker tissue and from soil in areas with high pitch canker disease incidence, and to several other species of Fusarium isolated from the same soil samples where Arthrobacter was recovered. Generally the pitch canker isolates were more sensitive to Arthrobacter than the soil fusaria. There was variation in the ability of the Arthrobacter isolates to inhibit the growth of the fusaria recovered from the soil at the three different test sites. Light and scanning electron microscope observations revealed that hyphae of FMS growing near an isolate of Arthrobacter were enlarged, producing many vesicularlike structures. The surface of these hyphae was warped and wrinkled in comparison with normal hyphae.

Antibiosis↗

PubMed1988

Speculative cell cycle in yeast-phase growth of Sporothrix schenckii: plasma membrane ultrastructure as revealed by freeze-fracture electron microscopy.

The cell cycle in yeast-phase growth of Sporothrix schenckii was investigated by light microscopy and freeze-fracture electron microscopy after a 3- to 7-day cultivation on brain heart infusion agar medium at 37 degrees C. Mother yeastlike cells were able to bear daughter yeastlike cells. They were also able to produce germ tubes that had the potential to develop into pseudohyphae and hyphae. On the other hand, hyphae or pseudohyphae born from yeastlike cells were able to bear yeastlike cells directly. These results lead us to propose a hypothetical cell cycle for yeast-phase growth involving yeastlike vegetative cells, pseudohyphae, and hyphae.

Cell Cycle↗

PubMed2000

Localization of fungal fimbriae by immunocytochemistry in pathogenic and nonpathogenic isolates of Venturia inaequalis.

Pathogenic and nonpathogenic isolates of Venturia inaequalis were grown in liquid culture. Hyphae were treated with two types of fimbrial antiserum (AU- and AV-1) and examined by immunofluorescent microscopy, in order to establish the distribution of fimbrial epitopes in whole cell mounts. The AV-1 antiserum was specific for the glycoprotein subunits while the AU-antiserum was specific for the protein moieties present on the fimbriae of Mycobotryum violaceum. The use of fimbrial antiserum with immunocytochemistry and transmission electron microscopy demonstrated a clear distinction between pathogenic and nonpathogenic isolates of V. inaequalis, based on the appearance of the fungal cell wall and the distribution of fimbrial epitopes labeled with AV-1 antiserum and immunogold complex. In actively growing hyphae of the pathogenic isolate, characterized by distinct cellular organelles, small vacuoles, and lipid bodies, fimbrial epitopes were concentrated in the fungal cell wall and were present minimally on the outer surface. In contrast, actively growing hyphae of the nonpathogenic isolate of V. inaequalis had extensive fine hair-like protrusions in the fungal cell wall which labeled with the AV-1 antiserum and immunogold. The distribution of fimbrial epitopes in V. inaequalis was highly dependent on the developmental growth stage of the fungal mycelium. Aging mycelia in both the pathogenic and nonpathogenic isolates of V. inaequalis were characterized by a large central vacuole and no label. In the pathogenic and nonpathogenic isolates of V. inaequalis grown in vitro, the distribution of fimbrial glycoprotein epitopes provided a more complex profile than that seen in M. violaceum.

Antibodies↗

PubMed1998

Evidence of self-inhibition by filamentous fungi accounts for unidirectional hyphal growth in colonies.

Hyphal growth in filamentous fungi proceeds in an unidirectional radial pattern from a point inoculation. An inhibitor produced, secreted, and absorbed by the advancing hyphae has been speculated to account for directional growth. Working with Mucor and Aspergillus, laboratory evidence is provided for the production of an inhibitory by the advancing hyphae of these filmentous fungi that precludes back growth. Strains of Mucor and Aspergillus inoculated in agar on a 0.45-mm membrane filter placed on agar surfaces grow radially beyond the filter perimeter and onto the agar surface. Removal of the filter pad does not result in hyphal extension back into the clear zone underlying the membrane filter. Inoculation of the clear zone with a fresh Mucor or Aspergillus spore suspension does not result in hyphal growth. Instead, germinating spores show aberrant and aborted hyphae. The fungal inhibitor shows cross-reactivity between Mucor and Aspergillus, is resistant to pronase (10 mg/mL) but not chloroform inactivation, and passes through dialysis tubing with a molecular weight cutoff of 3500.

Agar↗

PubMed1996

Development of murine monoclonal antibodies for the immunohistochemical diagnosis of systemic bovine aspergillosis.

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and -4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and -2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions (n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and -2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

Animals↗

PubMed2004

Polyadenylation of ribosomal RNA by Candida albicans also involves the small subunit.

BACKGROUND: Candida albicans is a polymorphic fungus causing serious infections in immunocompromised patients. It is capable of shifting from yeast to germinating forms such as hypha and pseudohypha in response to a variety of signals, including mammalian serum. We have previously shown that some of the large 25S components of ribosomal RNA in Candida albicans get polyadenylated, and this process is transiently intensified shortly after serum exposure just prior to the appearance of germination changes. RESULTS: We now present data that this process also involves the small 18S subunit of ribosomal RNA in this organism. Unlike the large 25S subunit, polyadenylation sites near the 3' end are more variable and no polyadenylation was found at the reported maturation site of 18S. Similar to 25S, one or more polyadenylated mature sized 18S molecules get intensified transiently by serum just prior to the appearance of hypha. CONCLUSIONS: The transient increase in polyadenylation of both the large and the small subunits of ribosomal RNA just prior to the appearance of hypha, raises the possibility of a role in this process.

Candida albicans↗

PubMed1994

Cytoskeletal regulation of ion channel distribution in the tip-growing organism Saprolegnia ferax.

Growing hyphal tips of the oomycete Saprolegnia ferax possess a tip-high gradient of stretch-activated ion channels permeable to calcium. These mechanosensitive channels appear to play a direct role in the polarized tip growth process. Treatment of S. ferax hyphae with cytochalasin E leads to the disruption of plasmalemma-associated, peripheral cytoplasmic actin populations and altered morphology of apical protoplasts, and eliminates the tip-high gradient of stretch-activated channels. Cytochalasin E did not alter the normal aggregation of stretch-activated channels. The density of spontaneous K+ channels was decreased in all regions of the hyphae after treatment with cytochalasin E. These results suggest that the peripheral F-actin network in the growing tip of S. ferax hyphae establishes or maintains the tip-high gradient of SA channels, either by the delivery of channel-bearing vesicles to the apex or by the interactions between the channels and the peripheral actin network.

Cytochalasins↗

PubMed2001

Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii.

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.

Actins↗

PubMed2001

Increasing diagnostic yield in allergic fungal sinusitis.

Diagnosis of allergic fungal sinusitis (AFS) in patients who present with rhinosinusitis and polyposis is based upon certain clinical, histopathological and mycological histopathological criteria are the demonstration of fungal hyphae in allergic mucin and absence of tissue invasion in the excised polyps. Previous reports have indicated difficulty in demonstrating fungal hyphae on histological examination in up to 75 per cent of cases. Analysis of a series of 25 patients with AFS, suggested methods to ensure demonstration of fungal hyphae and thus increase diagnostic yield in cases with suspected AFS. criteria. Specific diagnostic

Coloring Agents↗

PubMed2005

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa.

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.

Catalytic Domain↗

PubMed2007

Increased culture recovery of Zygomycetes under physiologic temperature conditions.

Poor recovery of Zygomycetes hyphae from tissue specimens may result from failure of current culture methods to mimic physiologic conditions found in hyphae-laden infected tissue. We describe the use of an in vitro model simulating Zygomycetes growth under necrotic or hypoxic tissue conditions. We preconditioned hyphae of clinical Zygomycetes isolates in flasks under anaerobic conditions using Ana-Packs (Becton Dickinson Microbiology Systems, Sparks, MD) at 37 degrees C for 48 hours, thus simulating in vivo growth in an infracted hypoxic lesion, and compared the recovery of paired inocula at 37 degrees C and 25 degrees C. Incubation of stock culture isolates at 37 degrees C resulted in significantly better culture recovery (about 10-fold) when compared with incubation at 25 degrees C (P < .0001). In addition, we similarly evaluated 25, 291 consecutive clinical specimens. Among 41 specimens, the yield of Zygomycetes cultures incubated at 37 degrees C (23/41 [56%]) was significantly higher than that incubated at 25 degrees C (9/41 [22%]; P = .0001). Overall, we found that culture recovery was significantly (254%) enhanced at 37 degrees C.

Anaerobiosis↗

PubMed2004

Antifungal treatment with carvacrol and eugenol of oral candidiasis in immunosuppressed rats.

Carvacrol and eugenol, the main (phenolic) components of essential oils of some aromatic plants, were evaluated for their therapeutic efficacy in the treatment of experimental oral candidiasis induced by Candida albicans in immunosuppressed rats. This anticandidal activity was analyzed by microbiological and histopathological techniques, and it was compared with that of nystatin, which was used as a positive control. Microbiologically, carvacrol and eugenol significantly (p<0.05) reduced the number of colony forming units (CFU) sampled from the oral cavity of rats treated for eight consecutive days, compared to untreated control rats. Treatment with nystatin gave similar results. Histologically, the untreated control animals showed numerous hyphae on the epithelium of the dorsal surface of the tongue. In contrast no hyphal colonization of the epithelium was seen in carvacrol-treated animals, while in rats treated with eugenol, only a few focalized zones of the dorsal surface of the tongue were occupied by hyphae. In the nystatin treated group, hyphae were found in the folds of the tongue mucosa. Thus, the histological data were confirmed by the microbiological tests for carvacrol and eugenol, but not for the nystatin-treated group. Therefore, carvacrol and eugenol could be considered as strong antifungal agents and could be proposed as therapeutic agents for oral candidiasis.

Animals↗

PubMed1996

Detection of Epstein-Barr virus early replicative phase using monoclonal antibody BZ-1 in oral hairy leukoplakia and other hyperkeratotic oral mucosal lesions: a retrospective study.

The early replicative phase of Epstein-Barr virus (EBV) was detected by immunohistochemistry, in addition to routine histology with hematoxylin eosin staining, in formalin fixed, paraffin-embedded archive specimens of 97 hyperkeratotic lesions including oral hairy leukoplakia (HL) in order to assess whether EBV is specific to HL, or if it occurs in other hyperkeratotic lesions of the oral mucosa. Monoclonal antibody (MAb) BZ-1 directed against the BZLF-1 gene product of EBV was used for immunostaining by the avidin-biotin peroxidase technique. The reaction was visualized with DAB/H2O2 solution and counterstained with PAS stain to detect any coexisting of hyphae of Candida. Eight lesions were reactive with BZ-1, confirming a diagnosis of HL, although the total number of lesions with koilocytoid features was 33. Seventy percent of the patients with confirmed HL lesions were HIV-seropositive, and 50% of the HL lesions were associated with Candida hyphae. One third of the lesions with koilocytoid features were associated with hyphae of Candida. Only one renal transplant patient had HL, confirmed by a positive BZ-1 reaction, although 60% of the hyperkeratotic lesions showed koilocytoid features in a subgroup of 15 patients with renal transplants.

AIDS-Related Opportunistic Infections↗

PubMed2002

Host-parasite relation in invasive aspergillosis.

Invasive aspergillosis has become one of the most important infectious complications of intensive modern medicine often limiting the success of oncological treatments and transplantation. The rationale for this is found in the effects of immunosuppressive therapies and to a lesser degree underlying disease processes as well as in properties of the fungus. The double pronged nature of host defenses directed against spores and hyphae of Aspergilli is affected by glucocorticoids and myeloablative therapy. Resident alveolar macrophages are the major players eliminating inhaled conidia. Neutrophils, efficiently killing hyphae. These defense mechanisms are so solid that even after the inhalation of billions of spores infection is reliably prevented in man. Mechanisms by which glucocorticoids prevent macrophages from first inhibiting germination of ingested conidia and then killing them have not been elucidated. Mobilization of neutrophils is also hampered by glucocorticoids that have the potential to suppress the expression of an array of neutrophil chemokines by macrophages. Glucocorticoids are thus able to abrogate defenses against Aspergilli on their own, affecting both lines of defense. In neutrophil granulocytes oxidative killing systems directed against hyphae are of major importance as pointed out by infections in children with chronic granulomatous disease, but other killing systems such as defensins and possibly thrombocidins are also of importance. To date it remains speculative that platelet derived thrombocidins play an important role, but such speculations are tempting in view of the angio-invasive nature of the fungus and the coexisting thrombocytopenia in many patients with aspergillosis.

Aspergillosis↗

PubMed2004

[A case of allergic fungal sinusitis caused by Bipolaris spicifera].

We describe a case of allergic fungal sinusitis (AFS) caused by Bipolaris spicifera, the first case reported in Japan. A 70-year-old man was admitted to our hospital because of diplopia following bilateral nasal obstruction and discharge. Radiological studies using computed tomographic scan showed a large soft tissue mass occupying the right frontal, bilateral ethmoid and sphenoid sinuses. He underwent drainage surgery and histopathological examination of the contents of the paranasal sinuses revealed scattered fungal hyphae within "allergic mucin". By cytological examination, these hyphae showed septation at irregular intervals, and were swollen to various sizes. Microbiological studies identified the fungus as Bipolaris spicifera. The presence of allergic mucin and scattered fungal hyphae were very important findings in making a diagnosis of AFS histopathologically, so squash cytology of the contents of the paranasal sinuses was quite useful to observe fungal elements and identify the strain in this case.

Aged↗

PubMed1989

Scanning electron microscopy of Malassezia furfur in tinea versicolor.

Morphologic features of Malassezia(M.) furfur in the horny layer from clinical lesions of tinea versicolor were examined by scanning electron microscopy and compared with the appearance of fungus in the horny layer from normal skin and in culture. In skin lesions of tinea versicolor, M. furfur showed a variety of growth and reproduction patterns. Although the main patterns were budding yeast forms, various patterns suggesting yeast-mycelial conversion were observed and mycelial hyphae were more prominent in the deeper horny layer than in the superficial layers. However, in the skin of normal persons and in culture, M. furfur existed only as yeast forms and no mycelial hyphae or yeast-mycelial conversion forms were seen. This suggests that the morphologic change of M. furfur, from a yeast form to a mycelial hypha one, may play a role in the induction of the clinical lesion of tinea versicolor.

Biopsy↗

PubMed2002

Dendritic cells pulsed with fungal RNA induce protective immunity to Candida albicans in hematopoietic transplantation.

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-gamma-producing CD4(+) donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.

Adoptive Transfer↗