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Helical growth of hyphae of Candida albicans.

When grown on a range of surfaces in conditions favouring hyphal growth, hyphae of Candida albicans grew in a right-handed helical fashion. This phenomenon was observed with eight strains and with two nutrient media. It is suggested that this is a result of rotation of the hyphal apex as it extends, which on some surfaces results in a helical hyphal wall, but which in a liquid results in a straight hypha. The consequence is that on a surface, a helically growing hypha will be exposed to a more diverse environment than a straight hypha. This phenomenon may have significance in the colonization of tissue by C. albicans.

Anti-Bacterial Agents↗

Inhibition of monocyte-mediated damage to fungal hyphae by steroid hormones.

Human monocytes can damage hyphal forms of C. albicans [1], R. oryzae [2], and A. fumigatus [3]. Because corticosteroids can inhibit phagocytosis and killing of bacteria by monocytes, the effects of steroid hormones on interactions of monocytes with hyphae were studied. Monocyte-mediated hyphal damage was inhibited by 10 microM hydrocortisone; inhibitory effects of lower concentrations of hydrocortisone were less marked with Rhizopus than with Candida or Aspergillus hyphae. Addition of 10% normal human serum to hyphae and monocytes did not affect the inhibitory effects of hydrocortisone (data not shown). Comparable inhibitory effects were obtained using 0.3-1.0 microM dexamethasone (data not shown). Estrogen and progesterone also inhibited damage to Candida or Rhizopus. However, inhibition occurred at concentrations which are readily achievable in vivo by pharmacologic doses of hydrocortisone, but not by other steroid hormones. Thus, pharmacologic doses of corticosteroids might produce serum hormone concentrations which could interfere with activity of host monocytes against the tissue-invasive forms of C. albicans and A. fumigatus in vivo. However, higher pharmacologic doses of these hormones may be required to inhibit leukocyte-mediated damage to Rhizopus hyphae. In contrast, predisposition of women to superficial candidal infections cannot be explained solely by direct effects of estrogens or progesterone on leukocyte-mediated hyphal damage.

Fungi↗

Unopsonized Candida albicans hyphae stimulate a neutrophil respiratory burst and a cytosolic calcium flux without membrane depolarization.

We have defined major differences between events elicited during activation of human neutrophils by opsonized Candida hyphae (noningestible) or zymosan (ingestible) and by unopsonized Candida hyphae (also noningestible). In response to opsonized particles, a transient increase in cytosolic free calcium was initially observed, followed by depolarization in the transmembrane potential and a respiratory burst. Without opsonins, zymosan did not elicit a response from neutrophils. In contrast, unopsonized Candida hyphae elicited a transient increase in cytosolic calcium, as well as triggered a respiratory burst; they did not, however, elicit a depolarization of the neutrophil membrane. Furthermore, the rate of superoxide generation decreased after a prolonged lag period. The calcium flux was also delayed and coincided with the onset of superoxide generation. Thus, our data demonstrate that Candida albicans hyphae activate neutrophils in the absence of added serum components and initiate a neutrophil respiratory burst without an antecedent membrane depolarization.

Calcium↗

Antifungal activity of elutriated human monocytes against Aspergillus fumigatus hyphae: enhancement by granulocyte-macrophage colony-stimulating factor and interferon-gamma.

Human monocytes are important effector cells in host defenses against Aspergillus hyphae, and as elutriated monocytes (EHM) they may be transfused in large quantities to leukopenic patients with invasive aspergillosis. The antifungal activity of EHM against Aspergillus hyphae was compared with that of polymorphonuclear leukocytes (PMNL). The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) on superoxide anion (O2-) release and on hyphal damage caused by EHM against unopsonized A. fumigatus hyphae was investigated. EHM had antihyphal activity comparable to that of PMNL. GM-CSF significantly augmented O2- release by EHM in response to PMA. Also, both GM-CSF and IFN-gamma significantly enhanced the antifungal activity of EHM compared with untreated controls. Thus, EHM have demonstrable antifungal activity against Aspergillus hyphae that may be increased by GM-CSF and IFN-gamma, suggesting their potential therapeutic role in immune reconstitution of effector cells.

Adult↗

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing.

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.

Cell Survival↗

The thn mutation of Schizophyllum commune, which suppresses formation of aerial hyphae, affects expression of the Sc3 hydrophobin gene.

The spontaneous and recessive mutation thn in the basidiomycete Schizophyllum commune suppresses the formation of aerial hyphae in the monokaryon and, if present as a double dose, the formation of both aerial hyphae and fruit-bodies in the dikaryon. In the monokaryon, the mutation prevents accumulation of mRNA of the Sc3 gene, and in the dikaryon it also prevents the accumulation of fruiting-specific mRNAs, including mRNAs of the Sc1 and Sc4 genes, which are homologous to the Sc3 gene. These three genes code for hydrophobins, a family of small hydrophobic cysteine-rich proteins. In the thn monokaryon, the only detectable change in synthesized proteins is the disappearance of an abundant protein of apparent Mr = 28 K from the culture medium and from the cell walls. Protein sequencing shows that this is the product of the Sc3 gene. The Sc3 hydrophobin is present in the walls of aerial hyphae as a hot-SDS-insoluble complex. Submerged hyphae excrete large amounts of the hydrophobin into the medium.

Amino Acid Sequence↗

Analysis of microtubules and F-actin structures in hyphae and conidia development of the opportunistic human pathogenic black yeast Aureobasidium pullulans.

Organization of the cytoskeleton was studied in the ascomycetous black yeast Aureobasidium pullulans, an opportunistic human pathogen, in an effort to present it as a potential target of antifungal therapy. Long cytoplasmic microtubules, extending along the hyphae from the base to the growing apex, were the dominant structures in multinucleate interphase cells. Before mitosis these microtubules disappeared and were replaced by intranuclear spindles. This reorganization of microtubules occurred along the whole length of hypha before synchronous division of the nuclei. Actin cytokinetic rings were rarely seen. Cortical actin in the form of patches accumulated in areas of cell wall growth, i.e. in the hyphal apex and near the occasionally formed septum. Actin cables were not seen. During synchronous conidiogenesis, the cytoplasmic microtubules extended along developing conidia, and actin patches lined their subcortical areas. Actin rings were formed regularly at the base of uninuclear conidia. Microtubule inhibitor methyl benzimidazol-2-ylcarbamate disintegrated the microtubules, and inhibited nuclear division, development of hyphae and conidiogenesis. Actin inhibitor Cytochalasin D induced swelling of hyphal apexes and developing conidia. This inhibitory activity ceased after 5 to 12 h when the occasional septa appeared and conidiogenesis was completed. The lack of unicellular organization in multinucleate hyphae of A. pullulans seems be related to a rarity of F-actin structures: i.e. absence of actin cables, the lack of actin cytokinetic rings in particular, resulting in the uncoupling of the nuclear division from cytokinesis; the association of both processes is, however, retained during conidiogenesis.

Actins↗

Effects of azole antifungal drugs on the transition from yeast cells to hyphae in susceptible and resistant isolates of the pathogenic yeast Candida albicans.

Oral infections caused by the yeast Candida albicans are some of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug resistance, creating a major problem in the treatment of yeast infections in AIDS patients and other immunocompromised individuals. Several molecular mechanisms that contribute to drug resistance have been identified. In C. albicans, the ability to morphologically switch from yeast cells (blastospores) to filamentous forms (hyphae) is an important virulence factor which contributes to the dissemination of Candida in host tissues and which promotes infection and invasion. A positive correlation between the level of antifungal drug resistance and the ability to form hyphae in the presence of azole drugs has been identified. Under hypha-inducing conditions in the presence of an azole drug, resistant clinical isolates form hyphae, while susceptible yeast isolates do not. This correlation is observed in a random sample from a population of susceptible and resistant isolates and is independent of the mechanisms of resistance. 35S-methionine incorporation suggests that growth inhibition is not sufficient to explain the inhibition of hyphal formation, but it may contribute to this inhibition.

Antifungal Agents↗

Studies of in vitro activities of voriconazole and itraconazole against Aspergillus hyphae using viability staining.

The minimal fungicidal concentrations (MFCs) of voriconazole and itraconazole for five clinical isolates each of Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger were determined by a broth macrodilution method. Conidial suspensions as inocula were compared to hyphae as inocula since the invasive form of aspergillosis is manifested by the appearance of hyphal structures. In addition, cell viability staining with the dye FUN-1 was performed to assess time-dependent damage of hyphae exposed to various concentrations of the antifungal agents. With conidial inocula the MFC ranges of voriconazole were 0.5 to 4 microg/ml and those of itraconazole were 0.25 to 2 microg/ml, whereas the MFCs (2 to >16 microg/ml) with hyphal inocula were substantially higher (P < 0.01) for both itraconazole and voriconazole. Only minor differences between the tested antifungals were observed since 16 of 20 and 17 of 20 of the isolates of Aspergillus spp. tested appeared to be killed by voriconazole and itraconazole, respectively. The results of FUN-1 viability staining correlated closely to colony counts, but various time- and dose-dependent levels of viability of hyphae were also observed. In conclusion, our study demonstrates the importance of the type of inoculum used to test antifungals and the applicability of FUN-1 staining as a rapid and sensitive method for assaying the viability of hyphae.

Antifungal Agents↗

Involvement of CD14 and toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae.

Invasive fungal infections represent an increasing problem associated with high mortality. The present study was undertaken to identify leukocyte subsets that are activated by hyphal fragments in a whole-human-blood model, as well as to examine the involvement of CD14 and Toll-like receptors (TLRs) in activation of monocytes by hyphae. Incubation of whole human blood with hyphal fragments from Aspergillus fumigatus and Scedosporium prolificans for 6 h caused induction of mRNAs for tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in T cells, B cells, and monocytes, but not in granulocytes, as analyzed by reverse transcription-PCR with mRNA isolated from very pure populations of these leukocyte subsets. In primary adherent human monocytes, induction of TNF-alpha by hyphal fragments was dependent on plasma. Heat treatment of plasma at 56 degrees C for 30 min strongly reduced the ability of plasma to prime for activation. Pretreatment of human monocytes with different concentrations (1, 3, and 10 microg/ml) of monoclonal antibody (MAb) HTA125 (anti-TLR4) or MAb 18D11 (anti-CD14) for 30 min inhibited the release of TNF-alpha induced by hyphal fragments in a dose-dependent manner. Maximal inhibitions of 35 and 70% were obtained with 10 microg of HTA125 and 18D11 per ml, respectively. In contrast, pretreatment with MAb TL2.1 (anti-TLR2) did not affect signaling induced by hyphae. Pretreatment with the lipid A antagonist B975 blocked lipopolysaccharide signaling but did not inhibit TNF-alpha production induced by hyphal fragments. Our results suggest that T cells, B cells, and monocytes are involved in the innate immune response to invasive fungal pathogens and that serum components are relevant for activation of monocytes by hyphae. CD14 and TLR4 may be involved in signaling of Aspergillus hyphae in monocytes, but further studies to elucidate this issue are warranted.

Aspergillus fumigatus↗

Age-dependent metabolic differences in peripheral hyphae of Rhizoctonia solani.

Peripheral hyphae were separated from the remaining thallus of Rhizoctonia solani in exponential and stationary phases of growth. The QO(2) in whole cells of peripheral hyphae from young fungal colonies was on the average 2.6 times and the protein content 1.6 times greater than in peripheral hyphae from old fungal colonies. The overall rate of amino acid uptake was less in old than in young fungal colonies. In a polyuridylic acid-polyphenylalanine incorporating system, the two kinds of peripheral hyphae required ribosomes, supernatant fraction, polyuridylic acid, soluble ribonucleic acid, adenosine triphosphate, and pyruvate kinase. The rate of polyphenylalanine synthesis in old fungal colonies was slower than in the young fungal colonies. The ribosomes and supernatant fraction of the young and old fungal colonies were interchangeable and active. The factor responsible for deficient protein synthesis in old fungal colonies appears to be in the soluble fraction of the mycelium.

Age Factors↗

Enzymes of ammonia assimilation in hyphae and vesicles of Frankia sp. strain CpI1.

Frankia spp. are filamentous actinomycetes that fix N2 in culture and in actinorhizal root nodules. In combined nitrogen-depleted aerobic environments, nitrogenase is restricted to thick-walled spherical structures, Frankia vesicles, that are formed on short stalks along the vegetative hyphae. The activities of the NH4(+)-assimilating enzymes (glutamine synthetase [GS], glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase) were determined in cells grown on NH4+ and N2 and in vesicles and hyphae from N2-fixing cultures separated on sucrose gradients. The two frankial GSs, GSI and GSII, were present in vesicles at levels similar to those detected in vegetative hyphae from N2-fixing cultures as shown by enzyme assay and two-dimensional polyacrylamide gel electrophoresis. Glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase activities were restricted to the vegetative hyphae. Vesicles apparently lack a complete pathway for assimilating ammonia beyond the glutamine stage.

Actinomycetales↗

Evaluation of miconazole activity contained in human serum to hypha of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic pathogen, especially in an immunocompromised host. This fungus grows in a hyphal form in infected tissues; therefore, new tests to examine hyphal susceptibility are needed. In this study, we measured the mycotic activity of miconazole (MCZ) contained in human serum against A. fumigatus using the BioCell-Tracer method. Three serum samples were obtained from the same patient who was injected with 600 mg b.i.d. MCZ daily for 2 days. The concentrations of MCZ in the serum sample were 8.8, 3.5, and 1.6 micro g/ml, respectively. The serum containing 8.8 micro g/ml of MCZ inhibited hyphal growth 90 minutes after administration, and the hypha stopped growing. The serum containing 3.5 micro g/ml MCZ stopped hypha growth 100 minutes after administration, but re-growth of the hypha was observed at this concentration of MCZ. Serum containing 1.6 micro g/ml did not inhibite hyphal growth, nor did control serum have any inhibitory activity foward hyphae. Based on these results, we conclude that the BioCell-Tracer is a useful method for determining the effects on filamentous fungi of antifungal agents in the serum.

Antifungal Agents↗

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae.

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51Cr release from radiolabeled monolayers. From these studies, we conclude that neutrophils are capable of killing Candida hyphae selectively within human vascular endothelial cell monolayers and may have protective rather than detrimental effects on endothelial cell integrity.

Candida albicans↗

Comparative study of the growth inhibition of vegetative hyphae of some dermatophytes and keratinophilic fungi on glucose-peptone-agar with griseofulvin.

By the method of Jesenská and Danilla [29] the growth inhibition of vegetative hyphae of colonies of 12 species of dermatophytes and keratinophilic fungi on the surface of glucose-peptone-agar with incorporated griseofulvin has been established. It could be concluded that the sensitivity of hyphae to griseofulvin is neither connected with the taxonomic position and ecological classification of these microscopic fungi, nor with their ability to survive in the soil, or with their pathogenity etc. - In Trichophyton rubrum some hyphae were found which were extremely resistant to griseofulvin. - The extablished values allow to make an objective comparison of the sensitivity level of vegetative hyphae of dermatophytes and keratinophilic fungi to griseofulvin in further geographical regions, at different times and in different laboratories. - In the discussion further possibilities in this direction are developed.

Arthrodermataceae↗

The fungal hydrophobin Sc3p self-assembles at the surface of aerial hyphae as a protein membrane constituting the hydrophobic rodlet layer.

The Schizophyllum commune hydrophobin Sc3p is a small, hydrophobic, cysteine-rich protein involved in the formation of aerial hyphae. Using an antibody against purified Sc3p we found that the hydrophobin is secreted into the medium at the apices of growing submerged hyphae but in emerging aerial hyphae it accumulates at the hyphal surface. Here, the hydrophobin self-assembles at the wall/air interface into an SDS-insoluble protein membrane, at the aerial site very hydrophobic and with the appearance of a mosaic of 10 nm spaced parallel rodlets. Interfacial self-assembly of the hydrophobin also occurs in vitro. When solutions containing various concentrations of purified Sc3p were dried down onto a glass surface, the amount of assembled Sc3p depended on the area of the interface. Surplus of Sc3p remained in the monomeric form, apparently because formation of a monolayer of assembled Sc3p abolishes the hydrophilic/hydrophobic interface. The 10 nm thick layer of assembled Sc3p at the surface of aerial hyphae thus probably represents a monolayer of the protein.

Animals↗

Incidental finding of Zygomyceteslike hyphae in the placenta. A case report.

BACKGROUND: Zygomycetes is a class of saprophytic fungi causing opportunistic infections. These fungi can cause six distinct clinical manifestations, which can be fatal without rapid diagnosis and treatment. The fungi have a predilection for blood vessel invasion, causing thrombosis, infarction and necrosis of the tissue. CASE: A 25-year-old black woman, a drug abuser, delivered a female infant and the placenta en route to the hospital. The estimated gestational age of the infant was 35 weeks. The infant and mother had an unremarkable hospital course. Evaluation of the placenta revealed extensive involvement of the membranes, umbilical cord and chorionic plate by fungal hyphae without any surrounding inflammation. These hyphae were seen invading blood vessels, but there was no evidence of thrombosis or necrosis. The morphology of the hyphae was consistent with Zygomycetes. The mother was contacted and claimed to be well. CONCLUSION: Only one case of placental involvement by Mucor has been published since 1966. Despite the observation of Zygomyceteslike hyphae in the placenta, both the mother and infant were reported to be doing well.

Adult↗

Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences.

Video-enhanced light microscopy of the apical and subapical regions of growing hyphae of several fungal species revealed the existence of momentary synchronized motions of subcellular organelles. First discovered in a temperature-sensitive morphological mutant (ramosa-1) of Aspergillus niger, these seemingly spontaneous cytoplasmic contractions were also detected in wild-type hyphae of A. niger, Neurospora crassa, and Trichoderma atroviride. Cytoplasmic contractions in all fungi lasted about 1 s. Although the cytoplasm recovered its motility and appearance, the contraction usually led to drastic changes in Spitzenkörper (apical body) behavior and hyphal morphology, often both. Within 10 s after the contraction, the Spitzenkörper commonly became dislodged from its polar position; sometimes it disassembled into phase-dark and phase-light components; more commonly, it disappeared completely. Whether partial or complete, the dislocation of the Spitzenkörper was always accompanied by a sharp reduction or cessation of growth, and was usually followed by marked morphological changes that included bulbous hyphal tips, bulges in the hyphal profile, and formation of subapical and apical branches. The cytoplasmic contractions are vivid evidence that the most conspicuous cell organelles (membrane-bound) in living hyphae are interconnected via a contractile cytoskeletal network.

Aspergillus niger↗