scholarly journals Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine

2012 ◽  
Vol 78 (24) ◽  
pp. 8623-8630 ◽  
Author(s):  
Rodrigo Almeida-Paes ◽  
Susana Frases ◽  
Glauber de Sousa Araújo ◽  
Manoel Marques Evangelista de Oliveira ◽  
Gary J. Gerfen ◽  
...  
Keyword(s):  
Culture Medium ◽  
Uv Light ◽  
Sporothrix Schenckii ◽  
Pigment Production ◽  
Yeast Cells ◽  
Fungal Cell ◽  
Content Type ◽  
Mutant Strains ◽  
Important Virulence Factor ◽  
Medical Interest

ABSTRACTSporothrix schenckiiis the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor ofS. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally,l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism inSporothrixspp., we cultured 73 strains, including representatives of newly describedSporothrixspecies of medical interest, such asS. brasiliensis,S. schenckii, andS. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. AnS. schenckiiDHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of theSporothrixcomplex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.

scholarly journals Altered Dynamics of Candida albicans Phagocytosis by Macrophages and PMNs When Both Phagocyte Subsets Are Present

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Fiona M. Rudkin ◽  
Judith M. Bain ◽  
Catriona Walls ◽  
Leanne E. Lewis ◽  
Neil A. R. Gow ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Immune System ◽  
Innate Immune System ◽  
Video Microscopy ◽  
Innate Immune ◽  
Live Cell ◽  
Yeast Cells ◽  
Polymorphonuclear Cells ◽  
Fungal Cell ◽  
Content Type

ABSTRACT An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection. IMPORTANCE Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.

scholarly journals FLO11Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

2013 ◽  
Vol 79 (19) ◽  
pp. 6023-6032 ◽  
Author(s):  
Marc Bou Zeidan ◽  
Lourdes Carmona ◽  
Severino Zara ◽  
Jose F. Marcos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Antimicrobial Peptides ◽  
Gene Knockout ◽  
Yeast Cells ◽  
Knockout Mutant ◽  
Fungal Cell ◽  
Content Type ◽  
Cell Penetrating ◽  
Flor Yeasts

ABSTRACTSaccharomyces cerevisiae“flor” yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends onFLO11gene length and expression.FLO11encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm ofS. cerevisiaeflor strains. We found that PAF26, a synthetic tryptophan-rich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functionalFLO11but was not accompanied by increasedFLO11expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that aflo11gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that theFLO11gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-to-cell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides.

scholarly journals Involvement of Fungal Cell Wall Components in Adhesion of Sporothrix schenckii to Human Fibronectin

2001 ◽  
Vol 69 (11) ◽  
pp. 6874-6880 ◽  
Author(s):  
Osana C. Lima ◽  
Camila C. Figueiredo ◽  
José O. Previato ◽  
Lucia Mendonça-Previato ◽  
Verônica Morandi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sporothrix Schenckii ◽  
Matrix Proteins ◽  
Yeast Cells ◽  
Binding Assays ◽  
Fungal Cell ◽  
Host Fungus ◽  
Human Fibronectin ◽  
Dose Dependent ◽  
Cell Surface Glycoconjugates

ABSTRACT Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable.S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca2+, inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.

scholarly journals Biosynthesis and Functions of Melanin inSporothrix schenckii

2000 ◽  
Vol 68 (6) ◽  
pp. 3696-3703 ◽  
Author(s):  
Rafael Romero-Martinez ◽  
Michael Wheeler ◽  
Antonieta Guerrero-Plata ◽  
Guadalupe Rico ◽  
Haydée Torres-Guerrero
Keyword(s):  
High Performance ◽  
Uv Light ◽  
Sporothrix Schenckii ◽  
Uv Spectra ◽  
Wild Type ◽  
Melanin Biosynthesis ◽  
Worldwide Distribution ◽  
Mutant Strains ◽  
In The Wild ◽  
Cutaneous Mycosis

ABSTRACT Sporothrix schenckii is a human pathogen that causes sporotrichosis, an important cutaneous mycosis with a worldwide distribution. It produces dark-brown conidia, which infect the host. We found that S. schenckii synthesizes melanin via the 1,8-dihydroxynaphthalene pentaketide pathway. Melanin biosynthesis in the wild type was inhibited by tricyclazole, and colonies of the fungus were reddish brown instead of black on tricyclazole-amended medium. Two melanin-deficient mutant strains were analyzed in this study: an albino that produced normal-appearing melanin on scytalone-amended medium and a reddish brown mutant that accumulated and extruded melanin metabolites into its medium. Scytalone and flaviolin obtained from cultures of the reddish brown mutant were identified by thin-layer chromatography, high-performance liquid chromatography, and UV spectra. Transmission electron microscopy showed an electron-dense granular material believed to be melanin in wild-type conidial cell walls, and this was absent in conidial walls of the albino mutant unless the albino was grown on a scytalone-amended medium. Melanized cells of wild-type S. schenckii and the albino grown on scytalone-amended medium were less susceptible to killing by chemically generated oxygen- and nitrogen-derived radicals and by UV light than were conidia of the mutant strains. Melanized conidia of the wild type and the scytalone-treated albino were also more resistant to phagocytosis and killing by human monocytes and murine macrophages than were unmelanized conidia of the two mutants. These results demonstrate that melanin protects S. schenckii against certain oxidative antimicrobial compounds and against attack by macrophages.

scholarly journals Functions of VPA1418 and VPA0305 Catalase Genes in Growth of Vibrio parahaemolyticus under Oxidative Stress

2016 ◽  
Vol 82 (6) ◽  
pp. 1859-1867 ◽  
Author(s):  
Ching-Lian Chen ◽  
Shin-yuan Fen ◽  
Chun-Hui Chung ◽  
Shu-Chuan Yu ◽  
Cheng-Lun Chien ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Culture Medium ◽  
Incubation Temperature ◽  
Content Type ◽  
Inhibition Of Growth ◽  
Homologous Genes ◽  
Mutant Strains ◽  
In The Wild ◽  
Incubation Temperatures ◽  
Logarithmic Growth

ABSTRACTThe marine foodborne enteropathogenVibrio parahaemolyticushas four putative catalase genes. The functions of twokatE-homologous genes,katE1(VPA1418) andkatE2(VPA0305), in the growth of this bacterium were examined using gene deletion mutants with or without complementary genes. The growth of the mutant strains in static or shaken cultures in a rich medium at 37°C or at low temperatures (12 and 4°C), with or without competition fromEscherichia coli, did not differ from that of the parent strain. When 175 μM extrinsic H2O2was added to the culture medium, bacterial growth of the ΔkatE1strain was delayed and growth of the ΔkatE1ΔkatE2and ΔkatE1ΔahpC1double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the ΔkatE1strain to the inhibition of growth by H2O2was higher at low incubation temperatures (12 and 22°C) than at 37°C. The determined gene expression of these catalase andahpCgenes revealed thatkatE1was highly expressed in the wild-type strain at 22°C under H2O2stress, while thekatE2andahpCgenes may play an alternate or compensatory role in the ΔkatE1strain. This study demonstrated thatkatE1encodes the chief functional catalase for detoxifying extrinsic H2O2during logarithmic growth and that the function of these genes was influenced by incubation temperature.

scholarly journals Chitinases Are Essential for Cell Separation in Ustilago maydis

2015 ◽  
Vol 14 (9) ◽  
pp. 846-857 ◽  
Author(s):  
Thorsten Langner ◽  
Merve Öztürk ◽  
Sarah Hartmann ◽  
Stefan Cord-Landwehr ◽  
Bruno Moerschbacher ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Separation ◽  
Morphological Changes ◽  
Ustilago Maydis ◽  
Yeast Cells ◽  
In Planta ◽  
Fungal Cell ◽  
Content Type ◽  
Chitinolytic Enzymes ◽  
Complete Life Cycle

ABSTRACTChitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such asUstilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a myceliumin planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity ofU. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

scholarly journals Cryptococcus neoformansCda1 and Its Chitin Deacetylase Activity Are Required for Fungal Pathogenesis

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Rajendra Upadhya ◽  
Lorina G. Baker ◽  
Woei C. Lam ◽  
Charles A. Specht ◽  
Maureen J. Donlin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogenesis ◽  
Mammalian Host ◽  
Infection Model ◽  
Chitin Deacetylase ◽  
Fungal Cell ◽  
Content Type ◽  
Mutant Strains

ABSTRACTChitin is an essential component of the cell wall ofCryptococcus neoformansconferring structural rigidity and integrity under diverse environmental conditions. Chitin deacetylase genes encode the enyzmes (chitin deacetylases [Cdas]) that deacetylate chitin, converting it to chitosan. The functional role of chitosan in the fungal cell wall is not well defined, but it is an important virulence determinant ofC. neoformans. Mutant strains deficient in chitosan are completely avirulent in a mouse pulmonary infection model.C. neoformanscarries genes that encode three Cdas (Cda1, Cda2, and Cda3) that appear to be functionally redundant in cells grown under vegetative conditions. Here we report thatC. neoformansCda1 is the principal Cda responsible for fungal pathogenesis. Point mutations were introduced in the active site of Cda1 to generate strains in which the enzyme activity of Cda1 was abolished without perturbing either its stability or localization. When used to infect CBA/J mice, Cda1 mutant strains produced less chitosan and were attenuated for virulence. We further demonstrate thatC. neoformansCda genes are transcribed differently during a murine infection from what has been measuredin vitro.IMPORTANCECryptococcus neoformansis unique among fungal pathogens that cause disease in a mammalian host, as it secretes a polysaccharide capsule that hinders recognition by the host to facilitate its survival and proliferation. Even though it causes serious infections in immunocompromised hosts, reports of infection in hosts that are immunocompetent are on the rise. The cell wall of a fungal pathogen, its synthesis, composition, and pathways of remodelling are attractive therapeutic targets for the development of fungicides. Chitosan, a polysaccharide in the cell wall ofC. neoformansis one such target, as it is critical for pathogenesis and absent in the host. The results we present shed light on the importance of one of the chitin deacetylases that synthesize chitosan during infection and further implicates chitosan as being a critical factor for the pathogenesis ofC. neoformans.

scholarly journals Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

2010 ◽  
Vol 9 (11) ◽  
pp. 1776-1787 ◽  
Author(s):  
Sabine Keppler-Ross ◽  
Lois Douglas ◽  
James B. Konopka ◽  
Neta Dean
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Early Recognition ◽  
Yeast Cells ◽  
Phagocytic Cells ◽  
Fungal Cell ◽  
Content Type ◽  
Murine Macrophages ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACT The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.

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.

scholarly journals Protein Phosphatase Pph3 and Its Regulatory Subunit Psy2 Regulate Rad53 Dephosphorylation and Cell Morphogenesis during Recovery from DNA Damage in Candida albicans

2011 ◽  
Vol 10 (11) ◽  
pp. 1565-1573 ◽  
Author(s):  
Ling Ling Sun ◽  
Wan Jie Li ◽  
Hai Tao Wang ◽  
Jie Chen ◽  
Ping Deng ◽  
...  
Keyword(s):  
Dna Damage ◽  
Candida Albicans ◽  
Uv Light ◽  
Pathogenic Fungus ◽  
Genotoxic Stress ◽  
Filamentous Growth ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Content Type ◽  
Dna Damaging Agents

ABSTRACT The ability of the pathogenic fungus Candida albicans to switch cellular morphologies is important for infection and virulence. Recent studies have revealed that C. albicans yeast cells can switch to filamentous growth under genotoxic stress in a manner dependent on the DNA replication/damage checkpoint. Here, we have investigated the functions of Pph3 (orf19.4378) and Psy2 (orf19.3685), whose orthologues in Saccharomyces cerevisiae mediate the dephosphorylation of the DNA damage checkpoint kinase Rad53 and the histone variant H2AX during recovery from DNA damage. Deleting PPH3 or PSY2 causes hypersensitivity to DNA-damaging agents, including cisplatin, methylmethane sulfonate (MMS), and UV light. In addition, pph3 Δ and psy2 Δ cells exhibit strong filamentous growth under genotoxic stress. Flow cytometry analysis shows that the mutant cells have lost the ability to adapt to genotoxic stress and remain arrested even after the stress is withdrawn. Furthermore, we show that Pph3 and Psy2 are required for the dephosphorylation of Rad53, but not H2AX, during DNA damage recovery. Taken together, these results show that C. albicans Pph3 and Psy2 have important roles in mediating genotoxin-induced filamentous growth and regulating Rad53 dephosphorylation.

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