Abstract

Understanding the molecular basis for survival in stress tolerant species goes hand in hand with the search for metabolites, compounds, and macromolecules playing a role in mechanisms of adaptation and being, at the same time, of possible biotechnological interest. The constant search for new products by the industry has indeed shifted the attention to extremophilic and extremotolerant organisms as potential producers of compounds with novel and unusual characteristics and functional activities under life-threatening conditions.

Reflecting this tendency, the objective of the present study is to deepen the knowledge on ecophysiology and systems biology of black fungi – a group of ascomycetes considered as among the most resistant Eukaryotes know to date – as well as to detect species possessing polymer degradation ability. Hence, the extremotolerant rock-associated species Knufia chersonesos and its nonmelanized spontaneous mutant, whose degradation skills have been revealed by preliminary studies, were chosen for a proteomic-based screening towards polyesterases.

Induced cultures – characterized by the addition of the biodegradable polyester poly(1,4-butylene adipate-coterephthalate) (PBAT) to the growth media – and control cultures were analysed by HPLC to determine the polymer hydrolysis. Both whole PBAT film and milled were tested. The induction was performed both in rich (2% malt extract, ME) and minimal medium (0.2% ME) aiming to test K. chersonesos ability to use PBAT as its sole carbon source. HPLC/MS identification and quantitation of the hydrolysis products terephtalic acid (Ta), mono(4-hydroxybutyl) terephthalate (BTa) and bis(4-hydroxybutyl) terephthalate (BTaB) indicated the presence of esters-hydrolyzing enzymes in the secretome of both strains and under induction with both whole and milled PBAT film. Ta was detected as the most abundant hydrolysis product, thus denoting degradation of PBAT to the smallest building block, especially in culture supernatants from minimal medium, resulting in up to 2-fold higher concentrations as compared to the other experimental conditions. Polymer hydrolysis was detected also when exposing un-induced growth media to the polymer, up to 70°C . Label-free shotgun proteomics and protein profiling showed largest differences in secretome composition and protein levels between minimal and reach growth medium. While at the optimal condition of growth an abundant and diverse set of proteins was detected growth on minimal medium lead to secretion of mostly carboxylic esterases.

Our results on the extracellular proteome of K. chersonesos demonstrate that the culture supernatant has hydrolytic ability when grown in standard media and when PBAT is added to the media. In conclusion, these analyses of polyester degrading activity show that the proteomic screenings of an organism’s extracellular proteome can aid the identification of novel polyesterases.

Abstract

Paracoccidioidomycosis (PCM) is a granulomatous systemic mycosis, whose etiological agents are dimorphic fungi of the genus Paracoccidioides. Melanin production by various fungi interferes in the mechanism of pathogenesis, and the same is observed in paracoccidioidomycosis. After analysis of melanin production by P. lutzii isolates (Pb01,Pb66, ED01,Pb1578and Pb8334) and P. brasiliensis isolates (Pb60855, Pb18 and Pbcão), we verified the ability of macrophages to phagocytose the highly virulent isolate Pb18, and the high and low producers of melanin isolates, Pb60855 and Pb01, respectively. Phagocytosis assay was carried out with C57BL/6 mice peritoneal macrophages that were challenged with antibodies/complement-treated or untreated yeast cells. Results showed that the presence of high concentrations of melanin reduced significantly the percentage of phagocytosed untreated Pb60855 and Pb18 yeast cells when compared to Pb01 isolate. SDS-PAGE protein and enzymatic profiles, including laccase activity, of the isolates Pb18, Pb60855 and Pb01 were also analyzed. The isolated Pb01 produced fewer proteins than Pb18 and Pb60855 in the tested conditions, as well as the laccase enzyme activity was reduced in isolate Pb01. Molecular phylogenetic studies have indicated two distinct clades among the genus Paracoccidioides: the lutzii clade containing P. lutzii species and the brasiliensis clade that harbors five phylogenetic cryptic species (S1a, S1b, PS2, PS3, and PS4) that were recently reclassified as formal species: P. americana (PS2), P. restrepiensis (PS3), P. venezuelensis (PS4) and P. brasiliensis sensu strictu (S1a and S1b). Our study included a representative isolates from each new formal species, that gives a differential production of melanin, lacasse, proteins and enzymatic profiles that could be explain the virulence among them. We conclude that the presence of lower concentrations of melanin and the reduced production of protein/enzymes by P. lutzii Pb01 isolate can be related to the augmented phagocytosis of these yeast cells by macrophages in vitro, explaining the reduced virulence of this isolate in vivo, in front of the other spices. Suggesting that there may be a differential expression of the virulence factors according to species that should be better studied.

Abstract

Melanins are complex polymers that are synthesized by members of all biological kingdoms, making them one of the great natural pigments. Despite the abundance of melanins in our planet's biomass, there remains a great deal of mystery surrounding this pigment. Fungi produce and utilize melanins in the enviroment and during disease conditions. Ongoing studies have revealed remarkable structural characteristics of this enigmatic pigment and have uncovered intreguing associations of melanin to fungal virulence. In this session, we will review what we know about fungal melanin and were this knowledge is taking us.

Abstract

Abstract

Abstract

Coloration affects the fitness of organisms, e.g. via changes in thermal properties. Although color has been extensively studied in animals and plants, the role of color in mushrooms, the reproductive organs of many fungi, is unknown. We use citizen-science data consisting of 739 European grid cells with 3,054 fungal species, 3.2 million observations, 29,490 color samples and a mega-phylogeny to show that mushroom assemblage color lightness increases with temperature, meaning that assemblages are darker in cold than in warm climates. Our findings suggest that thermal adaptation via dark mushrooms facilitates reproduction of fungal species and the maintenance of populations that drive carbon and nutrient cycling in cold environments. We thus propose the ‘thermal pigmentation hypothesis’, stating a thermal-adaptation of mushroom-forming fungi.