Immunoprotection against Cryptococcosis Offered by Znf2 Depends on Capsule and the Hyphal Morphology

Cryptococcosis claims close to 200,000 lives annually. There is no vaccine clinically available for this fungal disease.

Melanin production and laccase mediated oxidative stress alleviation during fungal-fungal interaction among basidiomycete fungi

Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. In the present study, we performed two sets of fungal-fungal interaction—Trametes coccinea (F3) with Leiotrametes lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) to understand the changes in the metabolite profile during the interaction process and how this process impacts the hyphal/mycelial morphology of the participating fungi. The metabolites produced during interaction of T. coccinea (F3) with L. lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) was analysed through liquid chromatography coupled to mass spectroscopy (LC-MS). Most of the metabolites secreted or produced during interaction are associated with defensive response. Further, visualization with scanning electron microscopy revealed that interaction between the tested fungi led to the changes in the hyphal morphology. The bipartite fungal interaction resulted in the production of a dark brown colour pigment—melanin as confirmed by the LC-MS, FTIR and NMR analysis. Moreover, the fungal–fungal interaction also led to increase in the production of laccase, a group of multicopper oxidases involved in detoxification of toxic compounds. Further, increased activity of superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide anion to hydrogen peroxide was also recorded during fungal–fungal interaction. Quantitative real-time PCR revealed upregulation of lcc1 (encoding a laccase enzyme) and few other stress related genes of T. versicolor during its hyphal interaction with T. coccinea, suggesting a direct correlation between laccase production and melanin production.

Competency of Clove and Cinnamon Essential Oil Fumigation against Toxigenic and Atoxigenic Aspergillus flavus Isolates

Aspergillus flavus is a frequent contaminant of maize grain. We isolated this fungus, determined the colony morphology and species (by internal transcribed spacer sequencing) and measured the aflatoxin content. The selected A. flavus fungi were placed into two groups, toxigenic and atoxigenic; both appeared similar morphologically, except that the atoxigenic group lacked sclerotia. An essential oil fumigation test with clove and cinnamon oils as antifungal products was performed on fungal conidial discs and fungal colonies in Petri plates. Cinnamon oil at 2.5 to 5.0 μL/plate markedly inhibited the mycelial growth from conidial discs of both strains, whereas clove oil showed less activity. The oils had different effects on fungal mycelia. The higher clove fumigation doses of 10.0 to 20.0 μL/plate controlled fungal growth, while cinnamon oil caused less inhibition. Compared with atoxigenic groups, toxigenic A. flavus responded stably. Within abnormal A. flavus hyphae, the essential oils degenerated the hyphal morphology, resulting in exfoliated flakes and shrinkage, which were related to fungal membrane injury and collapse of vacuoles and phialide. The treatments, especially those with cinnamon oil, increased the electroconductivity, which suggested a weak mycelium membrane structure. Moreover, the treatments with essential oils reduced the ergosterol content in mycelia and the aflatoxin accumulation in the culture broth. The fumigations with clove and cinnamon oils inhibited the development of both conidia and colonies of A. flavus in dose-dependent manners.

The GPI-Anchored GH76 Protein Dfg5 Affects Hyphal Morphology and Osmoregulation in the Mycoparasite Trichoderma atroviride and Is Interconnected With MAPK Signaling

The fungal cell wall is composed of a cross-linked matrix of chitin, glucans, mannans, galactomannans, and cell wall proteins with mannan chains. Cell wall mannans are directly attached to the cell wall core, while the majority of mannoproteins is produced with a glycosylphosphatidylinositol (GPI) anchor and then transferred to β-1,6-glucan in the cell wall. In this study, we functionally characterized the transmembrane protein Dfg5 of the glycoside hydrolase family 76 (GH76) in the fungal mycoparasite Trichoderma atroviride, whose ortholog has recently been proposed to cross-link glycoproteins into the cell wall of yeast and fungi. We show that the T. atroviride Dfg5 candidate is a GPI-anchored, transmembrane, 6-hairpin member of the GH76 Dfg5 subfamily that plays an important role in hyphal morphology in this mycoparasite. Alterations in the release of proteins associated with cell wall remodeling as well as a higher amount of non-covalently bonded cell surface proteins were detected in the mutants compared to the wild-type. Gene expression analysis suggests that transcript levels of genes involved in glucan synthesis, of proteases involved in mycoparasitism, and of the Tmk1 mitogen-activated protein kinase (MAPK)-encoding gene are influenced by Dfg5, whereas Tmk3 governs Dfg5 transcription. We show that Dfg5 controls important physiological properties of T. atroviride, such as osmotic stress resistance, hyphal morphology, and cell wall stability.

Cyclophilin BcCyp2 Regulates Infection-Related Development to Facilitate Virulence of the Gray Mold Fungus Botrytis cinerea

Cyclophilin (Cyp) and Ca2+/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca2+/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.

Melanin Production and Laccase Mediated Oxidative Stress Alleviation During Fungal-fungal Interaction Among Basidiomycetes Fungi

Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. In the present study, we performed two sets of fungal-fungal interaction - Trametes coccinea (F3) with Leiotrametes lactinea (F9) and Trametes coccinea (F3) with Trametes versicolor (F1) to understand the changes in the metabolite profile during the interaction process and how this process impacts the hyphal/mycelial morphology of the participating fungi. The metabolites produced during interaction of Trametes coccinea (F3) with Leiotrametes lactinea (F9) and Trametes coccinea (F3) with Trametes versicolor (F1) was analysed through Liquid Chromatography coupled to Mass Spectroscopy (LC-MS). Most of the metabolites secreted or produced during interaction are associated with defensive response. Further, visualization with scanning electron microscopy revealed that interaction between the tested fungi led to the change in the hyphal morphology of the one participating fungus. The bipartite fungal interaction resulted in the production of a dark brown colour pigment – melanin as confirmed by the LC-MS, FTIR and NMR analysis. Moreover, the fungal-fungal interaction also led to increase in the production of laccase, a group of multicopper oxidases involved in detoxification of toxic compounds. Further increased activity of superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide anion to hydrogen peroxide was also recorded during fungal– fungal interaction. Quantitative real-time PCR revealed upregulation of lcc1 (encoding a laccase enzyme) and few other stress related genes of T. versicolor during its hyphal interaction with T. coccinea, suggesting a direct correlation between laccase production and melanin production.

The effects of external Mn2+ concentration on hyphal morphology and citric acid production are mediated primarily by the NRAMP-family transporter DmtA in Aspergillus niger

Background Citric acid, a commodity product of industrial biotechnology, is produced by fermentation of the filamentous fungus Aspergillus niger. A requirement for high-yield citric acid production is keeping the concentration of Mn2+ ions in the medium at or below 5 µg L−1. Understanding manganese metabolism in A. niger is therefore of critical importance to citric acid production. To this end, we investigated transport of Mn2+ ions in A. niger NRRL2270. Results we identified an A. niger gene (dmtA; NRRL3_07789), predicted to encode a transmembrane protein, with high sequence identity to the yeast manganese transporters Smf1p and Smf2p. Deletion of dmtA in A. niger eliminated the intake of Mn2+ at low (5 µg L−1) external Mn2+ concentration, and reduced the intake of Mn2+ at high (> 100 µg L−1) external Mn2+ concentration. Compared to the parent strain, overexpression of dmtA increased Mn2+ intake at both low and high external Mn2+ concentrations. Cultivation of the parent strain under Mn2+ ions limitation conditions (5 µg L−1) reduced germination and led to the formation of stubby, swollen hyphae that formed compact pellets. Deletion of dmtA caused defects in germination and hyphal morphology even in the presence of 100 µg L−1 Mn2+, while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn2+ concentration. Growth of both the parent and the deletion strains under citric acid producing conditions resulted in molar yields (Yp/s) of citric acid of > 0.8, although the deletion strain produced ~ 30% less biomass. This yield was reduced only by 20% in the presence of 100 µg L−1 Mn2+, whereas production by the parent strain was reduced by 60%. The Yp/s of the overexpressing strain was 17% of that of the parent strain, irrespective of the concentrations of external Mn2+. Conclusions Our results demonstrate that dmtA is physiologically important in the transport of Mn2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow.