Expanding the Toolbox for Genetic Manipulation in Pseudogymnoascus: RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in P. verrucosus.

Fungi belonging to the genus Pseudogymnoascus have garnered increasing attention in recent years. One of the members of the genus, P. destructans, has been identified as the causal agent of a severe bat disease. Simultaneously, the knowledge of Pseudogymnoascus species has expanded, in parallel with the increased availability of genome sequences. Moreover, Pseudogymnoascus exhibits great potential as a producer of specialized metabolites, displaying a diverse array of biological activities. Despite these significant advancements, the genetic landscape of Pseudogymnoascus remains largely unexplored due to the scarcity of suitable molecular tools for genetic manipulation. In this study, we successfully implemented RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption in Pseudogymnoascus, using an Antarctic strain of Pseudogymnoascus verrucosus as a model. Both methods were applied to target azpA, a gene involved in red pigment biosynthesis. Silencing of the azpA gene to levels of 90% or higher eliminated red pigment production, resulting in transformants exhibiting a white phenotype. On the other hand, the CRISPR/Cas9 system led to a high percentage (73%) of transformants with a one-nucleotide insertion, thereby inactivating azpA and abolishing red pigment production, resulting in a white phenotype. The successful application of RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption represents a significant advancement in Pseudogymnoascus research, opening avenues for comprehensive functional genetic investigations within this underexplored fungal genus.

In vitro inhibition of Saccharomyces cerevisiae growth by Metschnikowia spp. triggered by fast removal of iron via two ways.

Simple and convenient innovative assays in vitro demonstrating Metschnikowia spp. competition with Saccharomyces cerevisiae for an essential nutrient iron are presented. The tested Metschnikowia strains possess a common genetically determined property of secreting a pulcherriminic acid which in the presence of iron (III) ions forms an insoluble red pigment pulcherrimin. Both initial accumulation in growing Metschnikowia cells and subsequent precipitation in the form of pulcherrimin in the media contribute to iron removal by functioning cells. The predominant way depends on the strain. Due to fast elimination of iron, the growth of S. cerevisiae can be inhibited by tested Metschnikowia strains at concentrations of elemental iron in the media not exceeding 12 mg kg-1. Inhibition can be regulated by additional supply of microquantities of iron onto the surface of the solid medium within 20-24 h. At relatively low concentrations of elemental iron (below 1 mg kg-1), additional supplements of iron onto the surface provide an advancement in understanding the inhibition possibilities and enable the assay control. Microscopy observations revealed that Metschnikowia chlamydospores are involved in iron removal at relatively high iron concentrations. The results may find application in development of new methodologies and strategies for biocontrol or inhibition of pathogenic microorganisms.

Pigment production by Fusarium solani BRM054066 and determination of antioxidant and anti-inflammatory properties.

The fungal kingdom has been widely studied as a source of bioactive compounds of interest to the pharmaceutical and food industry. This paper studies the production of natural red pigments by Fusarium solani BRM054066 in the submerged fermentation system, using Doehlert experimental design to determine optimal cultivation conditions. The chemical composition of the red pigment was determined by Nuclear Magnetic Resonance spectroscopy (NMR) and Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Antioxidant activity was assessed by the ability to sequester of free radical DPPH. In the analysis of anti-inflammatory activity, murine peritoneal macrophages activated by LPS were used, and the gene expression of TNF-α, IL-1ß, IL-6, IL-10 and IL-17 was determined using qPCR. As a result, it was found that agitation at 200 rpm and glucose concentration ≥ 20 g/L promote the best results in the production of red pigment. The chemical compounds identified were two naphthoquinones, fusarubin and dihydrofusarubin, and an anthraquinone, a bostrycoidin, being fusarubin the majority compound. The red pigment showed antioxidant activity by scavenge 50% of the DPPH radical, in a concentration of 24 µg/mL. The pigment also showed an effective anti-inflammatory capacity by reducing the overexpression of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 and promoting the production of anti-inflammatory IL-10 and IL-17, in murine macrophages activated by LPS (p < 0.05). According to the results, the fungus F. solani BRM054066, under optimized conditions of cultivation, proved to be a promising source of biologically active natural pigments with wide industrial applicability.

Granulomatous reaction to red tattoo pigment treated with allopurinol.

Granulomatous reactions to tattoo ink are most commonly associated with mercury sulfide, a component of red pigments. Treatment options show limited results. Allopurinol, an inhibitor of xanthine oxidase, has been reported as a successful alternative treatment to granulomatous disorders, such as sarcoidosis and granulomatous reactions to fillers and tattoos. We report a case of granulomatous reaction to red tattoo pigment treated with allopurinol for 6 months. Good clinical improvement could be noticed during this time. Two months after we stopped the treatment, the lesion recurred. Allopurinol emerges as an important drug for the management of granulomatous reactions caused by tattoo pigments. Based on the significant clinical improvement noticed during its use, we recommend new studies to elucidate all the potential benefits of the use of allopurinol for the treatment of granulomatous reactions to tattoo ink.

Prodigiosin found in Serratia marcescens y2 initiates phototoxicity in the cytomembrane

Background: Light can be absorbed by bacterial pigment and affects its growth. Prodigiosin is a red pigment found in various bacterial species. The purpose of this study was to investigate the impacts of light on prodigiosin production, biomass formation, and membrane integrity of Serratia marcescens y2. Results: S. marcescens y2 grew better and produced more intracellular prodigiosin in darkness than in illumination. The pigment leakage ratio from cells was detected more in light than in darkness conditions. Ethidium bromide uptake assay could visually prove the prodigiosin-related loss of membrane integrity under illumination. A higher concentration of malondialdehyde (MDA) was detected in light-treated culture than in darkness. Tests of different light treatments (red, yellow, blue and green) showed that the maximum extracellular pigment and the minimum biomass formation and intracellular pigment were obtained in green light. Conclusions: Prodigiosin could absorb light, and then initiate phototoxicity damage of the cytomembrane.