Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

The fine structure of the microconidium of blastomyces dermatitidis

Aspects of the fine structure of the microconidium of the mycelial phase of the dimorphic fungal pathogenBlastomyces dermatitidis as seen by techniques of scanning and transmission electron microscopy are described and illustrated by electron micrographs. The conidia ofB. dermatitidis undergo changes in the ultrastructural appearance of the cell wall as the spore matures. The cell wall becomes irregular in its thickness and possesses two distinct layers. No discrete or unique surface spines or projections were evident when the conidium ofB. dermatitidis was viewed by scanning electron microscopy. Upon maturity there is a marked deposition of lipid material in large, multiple storage bodies which occupy much of the cytoplasmic area. However, the cytoplasmic organelles appear to retain their structural integrity.     

Specific fluorescent antiglobulins for the detection and identification of blastomyces dermatitidis yeast-phase cells

Summary Two lots of rabbit anti-Blastomyces dermatitidis globulins were conjugated with fluorescein isothiocyanate. These reagents stained brightly elements of the yeast and mycelial phases of 10 strains ofB. dermatitidis. In addition, the labeled antibodies cross-reacted with elements of the yeast and mycelial phases of 7 strains ofHistoplasma capsulatum and cells of numerous other heterologous fungi. Adsorption of one lot of labeled antibodies twice with yeast cells ofH. capsulatum and once with elements ofGeotrichum candidum rendered the conjugate specific for the yeast phase ofB. dermatitidis. Three adsorptions with yeast cells ofH. capsulatum followed by a single adsorption with elements ofG. candidum rendered the second conjugate specific for yeast-phase cells ofB. dermatitidis. The specific reagents did not react with the mycelial phase of this fungus.     

Primary cutaneous cryptococcosis

Cottonseed protein agar and a modified Tween-albumin casein hydrolysate (TAC) medium were compared for the yeast phase conversion of Blastomyces dermatitidis strains including fresh isolates as well as strains maintained in long-term storage. It was found that both media converted all the B. dermatitidis (mycelial phase) strains studied to yeast phase in three days. The TAC medium has the added advantage that it is clear and the growth can be recognized earlier than in the opaque cottonseed agar medium. The conversion in most cases was more than 95% and the morphology of the yeast cells was uniformly typical with broad base budding. There was a striking difference between the sensitivity of the yeast and mycelial phases of B. dermatitidis strains. The yeast phase was usually more sensitive to Amphotericin B than the mycelial phase of B. dermatitidis. Similarly, the yeast phases of four out of six strains were more sensitive to ketoconazole than their respective mycelial phases, while two strains showed identical sensitivity in cottonseed agar. The yeast phase organism was more susceptible to Amphotericin B when cottonseed medium was used whereas the yeast phase showed more susceptibility to ketoconazole in TAC medium. Since the sensitivity among the various strains differed, it is necessary to determine the antifungal susceptibility of the pathogenic phase of the organism for initiating proper therapy and monitoring effectiveness.Dr. Rose actively participated in this research; expired February 2, 1984.     

Fine Structure and Spore Germination in Fusarium culmorum

The fine structure of the macroconidium of Fusarium culmorum(W. G. Smith) Sacc. is described. The presence of a mucilaginouscoat around the conidium and its expansion during germinationare confirmed. Studies of germinating conidia have shown changesin the numbers of organelles. The germ tube is shown to be formedfrom an entirely new wall laid down in the conidium, and theemergence of the germ tube is brought about by lysis of theoriginal condial wall.     

The influence of medium on the chemical composition ofBlastomyces dermatitidis

Whole cells and cell walls of the mycelial and yeast forms ofBlastomyces dermatitidis grown in four different media were analyzed for differences in lipid, fatty acid, carbohydrate, and protein contents. The bound (saponifiable) fatty acids of yeast and mycelial whole cells (but not the cell walls) vary considerably in response to growth medium. The percentage of readily extractable lipid varied somewhat in whole cells. The percentage of carbohydrate and protein of whole cells and cell walls are little affected by the medium in which the cells are grown.     

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

Protein Profiling of the Dimorphic Pathogenic Fungus, Sporothrix schenckii

Sporotrichosis is a common cutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii, which exhibits a temperature-dependent dimorphic switch. At 25°C, it grows in a mycelial phase, while at 37°C, it forms unicellular yeast cells. The formation of yeast cells was thought to be a requisite for the pathogenicity of S. schenckii. To identify fragments that might be related to morphogenesis, whole-cell proteins from the mold and early yeast stages of S. schenckii were analyzed using 2DE. Among thousands of protein molecules displayed, more than 300 showed a differential expression between the two phases. In particular, 24 yeast-specific proteins were identified using MALDI-TOF/MS. One of the most interesting proteins was a hybrid histidine kinase, DRK1, a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum that was abundant in the yeast phase. Our study introduced a new approach to study dimorphism in S. schenckii, and the data may help us better understand the molecular mechanisms of phase transition.     

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.     

The fine structure of Culicinomyces clavisporus invading mosquito larvae

Electron microscope observations were made of the Australian and U.S. strains of Culicinomyces clavisporus infecting mosquito larvae. The wall of the conidium is composed of an inner (primary) layer, an outer (secondary) layer, and an exterior coating of a mucopolysaccharide substance believed responsible for conidial adhesion to the host cuticle prior to germination and penetration. In some instances the wall of the conidium is ruptured during germination and new wall layers and mucoid coating form around the germ tube whereas in other specimens the conidial wall layers extend around the germ tube without fracturing. The most common invasion site is through the larval foregut following ingestion of conidia. The apex of the germ tube presses tightly against the surface of the foregut cuticle and the mucilaginous coating is stripped away. There is evidence to suggest that the host epicuticle, which disappears across the zone of contact with the germ tube, is utilized for nutrition of the invading fungus. A collar of cuticle forms around the germ tube apex and a narrow penetrant hyphae extends into the procuticle. It is believed that cuticular penetration is primarily enzymatic assisted by mechanical pressure. The penetrant hypha swells into an oval cell in the hypodermal region and vegetative hypha then invade the hemocoel. The cells of the hypodermis develop signs of degeneration presumably due to the secretion of toxic substances from the invading hyphae. Host reactions, involving melanization of the host tissues, are sometimes evident among the invading penetrant hyphae in the cuticle or in the hypodermal cells in contact with the fungus. Melanized capsules form around some of the hyphae within the hemocoel. These latter reactions do not directly involve host blood cells and are examples of “humoral encapsulation” similar to that described by other authors during invasion of pathogenic organisms into mosquito larvae and chironomid larvae.     

Studies on the molecular ecology of Blastomyces dermatitidis

The microecology of Blastomyces dermatitidis, the dimorphic etiologic agentof the potentially fatal systemic fungal infection, blastomycosis, is not well defined.Blastomyces dermatitidis may occur periodically at natural sites, perhaps aided by rotting organic material, animal droppings and physical changes. Semi-quantitative growth studies of B. dermatitidis on 2% agar plates determined the ability toutilize or tolerate a variety of substrates including simple and complex molecules as carbon source, and organic and inorganic nitrogen sources. Allantoin, creatinine, quanidoacetic acid, guanidine and cysteine may be used as sole nitrogen source. Allantoin in combination with dextrose, glycerol, lichenen, celloboise and other wood by-products support growth of B. dermatitidis at room temperature. The nutritional conversion of the fungus to the yeast form at room temperature, well demonstrated on allantoin/glycerol/yeast extract media, appears to be affected by certain inorganic compounds. The organism tolerates low to moderate levels of alpha-pinene, tannic acid, shikimic acid, veratryl alcohol, vanillic acid, and polyethyleneglycol-200. There are significant differences among isolates regarding growth on various substances at 20° and 37° centigrade. It appears that a variety of wood by-products and animal waste substrates, in combination, support the growth of B. dermatitidis. Their role in the ecological niche of B. dermatitidis, and the importance of nutritional dimorphism in the natural environment warrants further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dimorphism-associated changes in plasma membrane H+-ATPase activity of Candida albicans

In situ plasma membrane H⁺-ATPase activity was monitored during pH-regulated dimorphism of Candida albicans using permeabilized cells. ATPase activity was found to increase in both the bud and germ tube forming populations at 135 min which coincides with the time of evagination. Upon reaching the terminal phenotype the mycelial form exhibited higher H⁺-ATPase activity as compared to the yeast form. At the time of evagination H⁺-efflux exhibited an increase. K⁺ depletion resulted in attenuated ATPase activity and glucose induced H⁺-efflux. The results demonstrate that ATPase may play a regulatory role in dimorphism of C. albicans and K⁺ acts as a modulator.Abbreviations PM Plasma membrane - pHi intracellular pH - Pi inorganic phosphorus - TET Toluene: Ethanol: Triton X-100     

Blastomycosis: Report of the first case from Alberta,Canada

The first known case of laboratory confirmed blastomycosis in Alberta occurred in 1970. The patient, who is believed never to have left Alberta, presented with of headaches, sore neck and impaired intellect. Initially, tuberculous or cryptococcal meningitis was suspected, but laboratory findings did not support the diagnosis. A fungus resembling Blastomyces dermatitidis was isolated from the venticular cerebrospinal fluid and lung at autopsy. A few yeast cells suggestive of B. dermatitidis were seen in lung and brain tissue sections. Initial attempts at in vitro conversion of the mycelial form of the isolate into yeast form on several enriched media were unsuccessful. The fungus gave ± to ++ reactions with B. dermatitidis specific conjugate by the direct fluorescent antibody technique, it was not pathogenic for mice and guinea pigs, and no asexual spores were produced in slide cultures. Further investigation indicated that the mycelial form of the fungus converted into its yeast form when an actively growing inoculum was used, although the yeast cells varied considerably in size. The yeast form produced disseminated infection in mice within 10 days. Exoantigenic analysis demonstrated an A antigen specific for B. dermatitidis, which revealed the identity of this organism as an atypical strain of B. dermatitidis.

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Zusammenfassung Der erste Fall in Blastomycosis von einem Laboratorium wurde 1970 in Alberta bestätigt. Der Patient — der wahrscheinlich Alberta nie verlassen hat — klagte über Kopfschmerzen, Genickschmerzen und zeigte beeinträchtigte Verstand. Anfänglich wurde TBC — oder cryptococcal meningitis vermutet, laborfunde unterstutzten jedoch nicht die Diagnose. Ein Fungus — ahnlich zum Blastomyces dermatitidis wurde von venticular cerebrospinal Flüssigkeit und von der Lunge nach der Autopsie isoliert. Wenige Hefezellen hinweisend auf B. dermatitidis wurden gewebschnitten von Lunge und Gehirn festgestellt. Anfängliche Versuche die isolierten mycel Form in die Hefe Form in vitro mit Hilfe verschiedener angereicherter Nahrboden was erfolglos. Der Fungus ergab in der Direct Fluorescent Antibody Technik. ± zu ++ Reaktion mit dem B. dermatitidis specifische Konjugate; es war nicht pathogen fur Mause und Meerschweinchen, asexual Sporen wurden in der objecttrage Kultur produziert. Weitere Untersuchungen zeigten, dass sich die mycel Form des Fungus in die Hefeform umwandelte wenn ein gut wachsendes Inoculum gebraucht wurde. Die Hefezellen zeigten jedoch beträchtliche Grossenunterschiede. Die Hefeform verursachte in Mauseneine generallisierte Infektion in inerhalb von 10 Tagen. Exoantigenik Analyse zeigte ein A Antigen, specifisch für B. dermatitidis, welches die Identität des Organismus als atypischen Stamm des B. dermatitidis aufwies.

     

Lipid synthesis during reinitiation of growth from stationary phase cultures of Candida albicans

Lipid synthesis has been studied in the dimorphic fungus Candida albicans. ¹⁴C-acetate incorporation into lipid material was used to measure new lipid synthesis in two cultures in which either yeast or mycelial growth was initiated from stationary phase yeast cells. When resuspended in fresh medium at 37 °C, cells resume growth and change morphology while at 30 °C cells resume budding growth. When resuspended at the appropriate temperature, both yeast and germ tube cultures immediately incorporated ¹⁴C-acetate into lipid material. The labeled lipid was more or less evenly divided between neutral and phospholipid. Phosphatidyl choline was the major phospholipid fraction and along with phosphatidyl ethanolamine accounted for 60–65 % of the total phospholipid. Lipid synthesis during growth initiation of either morphology showed a similar pattern, with no significant differences observed in neutral or phospholipid or phospholipid components between yeast and mycelial forms.     

Importance of some factors on the dimorphism of Candida albicans

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal syntehtic medium of Shepherd et al. [19] modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.     

Nuclear behavior during the formation of appressoria byAlternaria alternata

Nuclear behavior in the developmental process of appressoria inAlternaria alternata was investigated. In pregerminated conidia, approximately 94% of the conidial cells were uninucleate. The migration of a nucleus into an elongating germ tube from a germinating conidium was confirmed after 2h of incubation at 24±1°C in PDB. Peak frequencies of binucleate and trinucleate germ tubes were detected 1 and 2h after the peak frequency of uninucleate germ tubes, respectively. Four-and five-nucleate germ tubes did not show marked peak frrequencies. A marked peak frequency of the six-nucleate germ tubes occurred about 1 h after the peak frequency of the trinucleate germ tubes, suggesting that the nuclei in the trinucleate germ tubes each divided once within 1 h. The significance of early establishment of multinucleate appressorial cells in the colonization of host plants by pathogenicA. alternata was discussed.     

Differential profiles of soluble proteins during the initiation of morphogenesis in Candida albicans

Candida albicans is a dimorphic fungus that can grow either as yeast or as mycelia. The mycelial form may be required for tissue penetration and therefore may have a role in pathogenesis. The protein profiles of the cell-free S100 fraction from budding yeast cells and germ tube-forming cells (an early stage of the transition between yeast and mycelia) were evaluated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yeast growth or germ tube formation was induced in carbon-starved cells at 37° C by either glucose, galactose or N-acetylglucosamine at pH 4.5 or pH 6.7. More than 400 constitutively synthesised polypeptides were identified on 2-D PAGE by silver staining. A few polypeptides which seem to reflect the release from carbon starvation were detected, but no polypeptides unique to either morphology were observed. Fractionation of S100 preparations by polyethylenimine or heparin-agarose affinity chromatography, which have been used to detect DNA-binding proteins, revealed several proteins that were synthesised on the resumption of cell growth or in response to pH difference. Heparin-agarose also bound novel polypeptides in the size range 130–200 kDa that were preferentially synthesised in germ tube-forming cells. These results suggest that any protein factors that might exert a regulatory role early in germ tube formation are of low abundance, and that a minor group of soluble proteins involved in C. albicans morphogenesis may be differentially synthesised. Received: 11 March 1996 / Accepted: 10 July 1996     

Protoplasts of systemic dimorphic fungal pathogens: Histoplasma capsulatum and blastomyces dermatitidis

Summary Protoplasts have been released fromH. capsulatum in the mycelial and yeast phases and from the mycelial and incompletly converted yeast phase ofB. dermatitidis by the enzymatic action of snail digestive juice. There is great variation in the mode of protoplast formation not only between species but between the two morphological forms, particularly inH. capsulatum These studies were supported in part by grants from the American Thoracic Socicty and the United States Public Health Service National Institute of Allergy and Infectious Diseases (1-R1-AI-7520-01).