scholarly journals Synthesis of Melanin-Like Pigments by Sporothrix schenckii In Vitro and during Mammalian Infection

2003 ◽  
Vol 71 (7) ◽  
pp. 4026-4033 ◽  
Author(s):  
Rachael Morris-Jones ◽  
Sirida Youngchim ◽  
Beatriz L. Gomez ◽  
Phil Aisen ◽  
Roderick J. Hay ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Proteolytic Enzymes ◽  
Sporothrix Schenckii ◽  
Brain Heart Infusion ◽  
Pathogenic Fungi ◽  
Yeast Cells ◽  
Important Virulence Factor ◽  
Mammalian Infection

ABSTRACT Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.

Evaluation of the in vitro and in vivo dimorphism ofSporothrix schenckii,Blastomyces dermatitidis, andParacoccidioides brasiliensisisolates after preservation in mineral oil

2004 ◽  
Vol 50 (6) ◽  
pp. 445-449 ◽  
Author(s):  
Renata Ferretti de Lima ◽  
Marcelly Maria dos Santos Brito ◽  
Guido Manoel Vidal Schäffer ◽  
Osana Cunha de Lima ◽  
Cintia de Moraes Borba
Keyword(s):  
Fungal Infections ◽  
Paracoccidioides Brasiliensis ◽  
Sporothrix Schenckii ◽  
Brain Heart Infusion ◽  
Morphological Differentiation ◽  
Pathogenic Fungi ◽  
Mineral Oil ◽  
Blastomyces Dermatitidis

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 °C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 °C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 °C and their micromorphology showed thin filaments with single hyaline conidia. At 36 °C the colonies did not differ from those grown at 25 °C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 °C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.Key words: pathogenic fungi, in vitro storage, in vivo passage, morphogenesis.

Melanization of Penicillium marneffei in vitro and in vivo

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 291-299 ◽  
Author(s):  
Sirida Youngchim ◽  
Roderick J. Hay ◽  
Andrew J. Hamilton
Keyword(s):  
Fungal Pathogens ◽  
Proteolytic Enzymes ◽  
Penicillium Marneffei ◽  
Yeast Cells ◽  
Resonance Spectroscopy ◽  
Stable Free Radical ◽  
Cell Extracts ◽  
Melanin Binding

Melanins are found universally in nature and are implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the conidia and the yeast cells of the thermally dimorphic fungal pathogen Penicillium marneffei produce melanin or melanin-like compounds in vitro and during infection. Treatment of conidia with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles that were similar in size and shape to the conidia. A melanin-binding monoclonal antibody (mAb) labelled pigmented conidia, yeast cells and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical compound, consistent with their identification as melanins. Skin tissue from penicilliosis marneffei patients contained yeast cells that were labelled by melanin-binding mAb. Additionally, sera from P. marneffei-infected mice developed a significant antibody response (both IgG and IgM) against melanin. Phenoloxidase activity capable of synthesizing melanin from l-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that P. marneffei conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that the yeast cells can synthesize pigment in vivo. Accordingly this pigment may play some role in the virulence of P. marneffei.

scholarly journals Detection of Melanin-Like Pigments in the Dimorphic Fungal Pathogen Paracoccidioides brasiliensis In Vitro and during Infection

2001 ◽  
Vol 69 (9) ◽  
pp. 5760-5767 ◽  
Author(s):  
Beatriz L. Gómez ◽  
Joshua D. Nosanchuk ◽  
Soraya Dı́ez ◽  
Sirida Youngchim ◽  
Philip Aisen ◽  
...  
Keyword(s):  
Fungal Pathogen ◽  
Proteolytic Enzymes ◽  
Paracoccidioides Brasiliensis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Yeast Cells ◽  
Resonance Spectroscopy ◽  
Microbial Infections ◽  
Melanin Binding

ABSTRACT Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium withl-3,4-dihydroxyphenylalanine (l-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in l-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from l-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

scholarly journals Melanization of Cryptococcus neoformans and Histoplasma capsulatum Reduces Their Susceptibilities to Amphotericin B and Caspofungin

2002 ◽  
Vol 46 (11) ◽  
pp. 3394-3400 ◽  
Author(s):  
David van Duin ◽  
Arturo Casadevall ◽  
Joshua D. Nosanchuk
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Clinical Laboratory ◽  
National Committee ◽  
Broth Macrodilution ◽  
Mammalian Infection

ABSTRACT The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of l-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with l-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.

scholarly journals Mechanism of Growth Regulation of Yeast Involving Hydrogen Sulfide From S-Propargyl-Cysteine Catalyzed by Cystathionine-γ-Lyase

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhongkai Gu ◽  
Yufan Sun ◽  
Feizhen Wu
Keyword(s):  
Hydrogen Sulfide ◽  
Growth Regulation ◽  
Biological Activities ◽  
Fungal Growth ◽  
Pathogenic Fungi ◽  
Signaling Molecules ◽  
Yeast Cells ◽  
Anti Fungal

Pathogenic fungi are recognized as a progressive threat to humans, particularly those with the immunocompromised condition. The growth of fungi is controlled by several factors, one of which is signaling molecules, such as hydrogen sulfide (H2S), which was traditionally regarded as a toxic gas without physiological function. However, recent studies have revealed that H2S is produced enzymatically and endogenously in several species, where it serves as a gaseous signaling molecule performing a variety of critical biological functions. However, the influence of this endogenous H2S on the biological activities occurring within the pathogenic fungi, such as transcriptomic and phenotypic alternations, has not been elucidated so far. Therefore, the present study was aimed to decipher this concern by utilizing S-propargyl-cysteine (SPRC) as a novel and stable donor of H2S and Saccharomyces cerevisiae as a fungal model. The results revealed that the yeast could produce H2S by catabolizing SPRC, which facilitated the growth of the yeast cells. This implies that the additional intracellularly generated H2S is generated primarily from the enhanced sulfur-amino-acid-biosynthesis pathways and serves to increase the growth rate of the yeast, and presumably the growth of the other fungi as well. In addition, by deciphering the implicated pathways and analyzing the in vitro enzymatic activities, cystathionine-γ-lyase (CYS3) was identified as the enzyme responsible for catabolizing SPRC into H2S in the yeast, which suggested that cystathionine-γ-lyase might play a significant role in the regulation of H2S-related transcriptomic and phenotypic alterations occurring in yeast. These findings provide important information regarding the mechanism underlying the influence of the gaseous signaling molecules such as H2S on fungal growth. In addition, the findings provide a better insight to the in vivo metabolism of H2S-related drugs, which would be useful for the future development of anti-fungal drugs.

scholarly journals Melanogenesis in dermatophyte species in vitro and during infection

Microbiology ◽  
2011 ◽  
Vol 157 (8) ◽  
pp. 2348-2356 ◽  
Author(s):  
Sirida Youngchim ◽  
Soraya Pornsuwan ◽  
Joshua D. Nosanchuk ◽  
Wiyada Dankai ◽  
Nongnuch Vanittanakom
Keyword(s):  
Fungal Pathogens ◽  
Proteolytic Enzymes ◽  
Agar Plate ◽  
Pathogenic Fungi ◽  
Trichophyton Mentagrophytes ◽  
Resonance Spectroscopy ◽  
Microsporum Gypseum ◽  
Stable Free Radical ◽  
Melanin Binding

Dermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.

scholarly journals Histoplasma capsulatum Synthesizes Melanin-Like Pigments In Vitro and during Mammalian Infection

2002 ◽  
Vol 70 (9) ◽  
pp. 5124-5131 ◽  
Author(s):  
Joshua D. Nosanchuk ◽  
Beatriz L. Gómez ◽  
Sirida Youngchim ◽  
Soraya Díez ◽  
Philip Aisen ◽  
...  
Keyword(s):  
Minimal Medium ◽  
Histoplasma Capsulatum ◽  
Proteolytic Enzymes ◽  
Pathogenic Fungi ◽  
Yeast Cells ◽  
Resonance Spectroscopy ◽  
Cell Extracts ◽  
Mouse Tissues ◽  
Melanin Binding

ABSTRACT Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogen Histoplasma capsulatum var. capsulatum produced melanin or melanin-like compounds in vitro and during infection. Growth of H. capsulatum mycelia in chemically defined minimal medium produced pigmented conidia. Growth of H. capsulatum yeast in chemically defined minimal medium with l-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected with H. capsulatum and from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance to H. capsulatum yeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that H. capsulatum conidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

scholarly journals Specific immunological test for the rapid identification of members of the genus Histoplasma

1976 ◽  
Vol 3 (2) ◽  
pp. 191-199
Author(s):  
P G Standard ◽  
L Kaufman
Keyword(s):  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Brain Heart Infusion ◽  
Pathogenic Fungi ◽  
Rapid Identification ◽  
In Vivo Studies ◽  
Immunological Test ◽  
Chrysosporium Keratinophilum ◽  
Culture Supernatants

A sensitive and specific immunological method was developed for rapid identification of the mycelial forms of Histoplasma capsulatum var. capsulatum, H. capsulatum var. duboisii, and H. farciminosum and for separation of these pathogenic fungi from morphologically similar hyphomycetes and other fungal pathogens. This method is based on the fact that all of the Histoplasma spp. produce H and M histoplasmin antigens, whereas the other fungi do not. Inocula consisting of heavy mycelial growth from a pure, full-grown culture were transferred into flasks containing small volumes of brain heart infusion broth. These cultures were placed on a shaker and grown at 25 C. Using the micro-immunodiffusion technique and antisera containing antibodies to H and M precipitinogens, we detected exoantigens in 3-day-old brain heart infusion culture supernatants concentrated 25 and 50 times. The ability of the procedure to identify Histoplasma spp. was evaluated by testing 96 unknown mycelial cultures that grossly or microscopically resembled Histoplasma spp. Three- and six-day-old concentrated culture supernatants prepared from each unknown were tested against rabbit anti-Arthroderma tuberculatum, Chrysosporium keratinophilum, H. capsulatum var. duboisii, and Corynascus (Thielavia) sepedonium sera and human histoplasmosis case serum. Each unknown was also identified by conventional laboratory procedures involving cultural and, where necessary, in vivo studies. In the comparative evaluation the immunological test was observed to be 100% sensitive. It permitted the accurate generic identification of the Histoplasma spp. within 5 days, in contrast to the average of 33 days required by the routine mycological procedure.

scholarly journals In Vitro and In Vivo Interactions of TOR Inhibitor AZD8055 and Azoles against Pathogenic Fungi

2022 ◽  
Author(s):  
Yi Sun ◽  
Lihua Tan ◽  
Zhaoqian Yao ◽  
Lujuan Gao ◽  
Ji Yang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Combination Therapy ◽  
Fungal Pathogens ◽  
Pathogenic Fungi ◽  
Valuable Alternative ◽  
Clinical Challenge

Limited options of antifungals and the emergence of drug resistance in fungal pathogens has been a multifaceted clinical challenge. Combination therapy represents a valuable alternative to antifungal monotherapy.

scholarly journals Assessing the bioactive profile of anti-fungal loaded calcium sulfate against fungal biofilms

2021 ◽  
Author(s):  
Mark C. Butcher ◽  
Jason L. Brown ◽  
Donald Hansom ◽  
Rebecca Wilson-van Os ◽  
Craig Delury ◽  
...  
Keyword(s):  
Calcium Sulfate ◽  
Fungal Pathogens ◽  
Pathogenic Fungi ◽  
Fungal Species ◽  
Antifungal Drugs ◽  
Release Mechanism ◽  
Local Antibiotics ◽  
Biofilm Inhibition

Calcium sulfate (CS) has been used clinically as a bone or void filling biomaterial, and due to its resorptive properties have provided the prospect for its use as a release mechanism for local antibiotics to control biofilms. Here, we aimed to test CS beads loaded with three antifungal drugs against planktonic and sessile fungal species to assess whether these antifungal beads could be harnessed to provide consistent release of antifungals at biofilm inhibitive doses. A panel of different fungal species (n=15) were selected for planktonic broth microdilution testing with fluconazole (FLZ), amphotericin B (AMB) and caspofungin (CSP). After establishing planktonic inhibition, antifungal CS beads were introduced to fungal biofilms (n=5) to assess biofilm formation and cell viability through a combination of standard quantitative and qualitative biofilm assays. Inoculation of a hydrogel substrate, packed with antifungal CS beads, was also used to assess diffusion through a semi-dry material, to mimic active infection in-vivo. In general, antifungals released from CS loaded beads were all effective at inhibiting the pathogenic fungi over 7-days within standard MIC ranges for these fungi. We observed a significant reduction of pre-grown fungal biofilms across key fungal pathogens following treatment, with visually observable changes in cell morphology and biofilm coverage provided by scanning electron microscopy. Assessment of biofilm inhibition also revealed reductions in total and viable cells across all organisms tested. These data show that antifungal loaded CS beads produce a sustained antimicrobial effect, which inhibits and kills clinically relevant fungal species in-vitro as planktonic and biofilm cells.

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