Hidden highways: fungi associated with glossiphoniid leeches

Optimal biodegradation of the herbicide haloxyfop-R-methyl by the fungus Mucor circinelloides YMM22

To establish a specific and sensitive TaqMan multiplex real-time quantitative PCR (RT-qPCR) assay to rapidly detect Mucorales. Customized oligonucleotide primers and corresponding detection probes were developed using the 18S ribosomal RNA gene sequences of Rhizomucor spp., Lichtheimia spp., and Mucor spp./Rhizopus spp. A multiplex RT-qPCR assay was established using TaqMan fluorescent probes, and its specificity, repeatability, and sensitivity were evaluated. The method was also used for the detection of simulated Mucorales-positive clinical samples and on samples from clinical patients possibly having fungal infections. The established TaqMan fluorescent probe-based multiplex RT-qPCR assay accurately detected Mucorales with high sensitivity, showing a minimum detection limit of 1fg/μL. The assay also showed high specificity and accurately detected organisms representing the four main genera in Mucorales (i.e., Rhizomucor miehei, Lichtheimia corymbifera, Mucor circinelloides, and Rhizopus oryzae). Additionally, no cross-reactivity was observed with 21 non-targeted strains (including viruses, bacteria, fungi, and Cunninghamella bertholletiae). The reproducibility of the assay was confirmed, with a relative standard deviation within groups of less than 1%. The assay was able to detect Mucorales pathogens (i.e., Rhizomucor spp., Lichtheimia spp., Mucor spp., and Rhizopus spp.) in simulated positive clinical specimens of blood, urine, lavage fluid, and sputum. Among 56 specimens from clinical patients with suspected fungal infections that were tested using the assay, one positive specimen was detected, consistent with the findings from sequencing and fungal culture identification. A highly specific and sensitive TaqMan fluorescent probe-based multiplex RT-qPCR detection method for Mucorales was established that has promising applications in the early and rapid diagnosis of Mucorales pathogens in clinical specimens.

Abstract Producing biolipids from different feedstocks has opened up new possibilities for renewable energy, these sources help address the rising global demand for energy. Micro‐organisms that store more than 20% lipids by dry cell weight are known as oleaginous, and their ability to build up large lipid reserves is important because they mainly store these as triacylglycerols. These lipids are not only vital for the microbes themselves, but also serve as key building blocks for biodiesel and other bioproducts. Researchers have focused on oleaginous micro‐organisms like Yarrowia lipolytica, Cryptococcus curvatus, Rhodotorula glutinis (yeasts), Mucor circinelloides (fungi), and microalgae such as Chlorella vulgaris and Nannochloropsis sp . because of their high lipid yields. This review examines recent progress in feedstocks, mechanisms, and bioprocesses that boost lipid production. We analyze current studies to identify promising yet less‐studied microbial strains and process improvements that could be applied on a larger scale. We also closely examine major challenges, including scaling up, reducing costs, complying with regulations, and securing public support. Finally, we suggest research steps, such as metabolic engineering, integrated biorefineries, and economic analysis, to help translate lab breakthroughs into real‐world bioenergy production. This review addresses the previous limitation and serves as roadmap for future research in sustainable microbial lipid production.

Marine bacteria are a valuable source of bioactive polysaccharides with therapeutic potential. In our previous study, we identified an exopolysaccharide produced by Kocuria sp. strain AG5 (EPSR5) isolated from the Red Sea, and demonstrated that it is a high-molecular weight, acidic heteropolysaccharide rich in sulfate (25.6%) and uronic acid (21.77%) groups. In the current study, we expanded the structural characterization and biological evaluation of EPSR5. GC–MS and HPLC analyses revealed glucose as the major monosaccharide, accompanied by xylose, arabinose, and glucuronic acid, confirming its heteropolysaccharide and polyanionic character. Thermal analysis (TGA) demonstrated stability up to 257 °C, while SEM–EDX revealed a porous, flake-like morphology enriched in oxygen, sodium, and phosphorus, suggesting phosphorylation and ionic associations. The biological activity of EPSR5 was assessed through a range of pharmacological assays. Antimicrobial testing against seven pathogenic microbes, including two fungal strains (Candida albicans, Mucor circinelloides), two Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus), and three Gram-negative bacteria (Escherichia coli, Salmonella typhi, Helicobacter pylori), demonstrated potent inhibitory effects. EPSR5 showed notable inhibition zones up to 30 mm against E. faecalis and 27 mm against H. pylori, with MIC/MBC values as low as 7.8/15.62 μg mL−1 and 15.62/15.62 μg mL−1, respectively. Significant antibiofilm activity was also observed, with inhibition rates reaching up to 96.54% against E. faecalis and 97.39% against H. pylori, in a concentration-dependent manner. Antioxidant potential was confirmed via TAC and FRAP assays, showing values of 62.8 ± 0.3 and 114.9 ± 0.5 μg ascorbic acid equivalents per mg, respectively. EPSR5 exhibited antidiabetic activity by inhibiting α-glucosidase and α-amylase enzymes with IC50 values of 17.06 μg mL−1 and 66.35 μg mL−1, respectively, comparable to Acarbose. Additionally, EPSR5 inhibited pancreatic lipase with an IC50 of 41.39 μg mL−1 and suppressed 90.1% of enzyme activity at 1000 μg mL−1. In wound healing assays, EPSR5 promoted fibroblast migration and accelerated wound closure, reducing the wound area within 48 hours, achieving 71.85% closure compared to 66.24% in control cells. Overall, these findings suggest that EPSR5 from Kocuria sp. AG5 exhibits multifaceted bioactivities with promising therapeutic potential for antimicrobial, antioxidant, antidiabetic, and wound healing applications.

Mucormycosis is a life-threatening infection caused by fungi of the Mucorales order, typically associated with immunocompromised hosts, but increasingly reported in immunocompetent individuals. This study investigated fungal burden, Th1/Th17 inflammatory profiles, and organ-specific dynamics in immunocompetent BALB/c mice intravenously infected with Rhizopus oryzae, Mucor circinelloides, or Rhizomucor pusillus. Colony-forming units were quantified in spleen, liver, and kidney at multiple time points, while serum cytokines and oxidative stress markers were analyzed. The results showed fungal persistence primarily in the spleen, accompanied by species-specific Th1/Th17 responses: R. oryzae induced the highest inflammatory response among all groups, with maximal cytokine production observed on day 7, particularly for IL-17A (352.58 pg/mL). In contrast, M. circinelloides exhibited its peak cytokine levels earlier, reaching the highest TNF-α concentration on day 3 (425.43 pg/mL). Meanwhile, R. pusillus triggered an early but moderate inflammatory response, with a maximum TNF-α value of 372.62 pg/mL detected on day 1, followed by clearance. Correlation analysis highlighted distinct immunological patterns, with IL-10 acting as a negative regulator of inflammation, while TNF-α and IL-17A reflected infection intensity depending on species and timing. The spleen emerged as a key organ coordinating immune responses during systemic infection. These findings reveal that mucormycosis in immunocompetent hosts triggers complex, species-dependent immune dynamics beyond classical immunosuppression, emphasizing the need to consider host–pathogen interactions when developing targeted antifungal strategies.

Silver nanoparticles (AgNPs) were synthesized using aqueous fruit extracts of Garcinia gummi-gutta (Gg) and Garcinia indica (Gi) via a phyto-mediated approach. Characterization by UV-Vis, FT-IR, XRD, DLS, zeta potential, EDS, and HR-TEM confirmed nanoscale dimensions (5-32nm), crystalline structure, negative surface charge, and phytochemical-mediated capping. GC-MS profiling of the extracts identified bioactive metabolites responsible for Ag⁺ reduction and nanoparticle stabilization. Antifungal activity was assessed against four mucormycosis-causing fungi-Absidia blakesleeana, Apophysomyces elegans, Mucor circinelloides, and Rhizopus arrhizus-using disc diffusion and minimum inhibitory concentration (MIC) assays. Gg-AgNPs showed significantly larger inhibition zones (16 ± 0.5mm for A. blakesleeana; 14 ± 0.33mm for M. circinelloides) than Gi-AgNPs (10 ± 0.33mm and 10 ± 0.5mm, respectively) (p < 0.001). MIC values were lower for Gg-AgNPs (15.625-62.5µg/mL), with A. elegans being the most susceptible. Synergistic assays revealed that Gg-AgNPs enhanced the efficacy of Amphotericin-B and Ketoconazole, increasing inhibition zones by up to 77% against A. elegans and 50% against A. blakesleeana. Statistical analyses (t test, two-way ANOVA) confirmed significant effects of nanoparticle type, fungal strain, and treatment modality (p < 0.001). These results demonstrate that Garcinia fruit extracts serve as efficient bio-reductants for sustainable nanoparticle synthesis, and the resulting AgNPs possess potent, statistically validated anti-mucormycotic activity. This comparative study highlights their potential as eco-friendly nanotherapeutics and synergistic adjuvants against mucormycosis pathogens.

The nitrogen regulator AreA modulates lipid metabolism through uga2 in Mucor circinelloides.

The filamentous fungus Mucor circinelloides IBT-83 is an excellent producer of intracellular lipids. This makes it a promising candidate for large-scale cultivation for industrial oil production. However, this process generates significant amounts of defatted biomass, which can serve as a source of valuable compounds such as chitosan. It was shown that almost 100 g of chitosan (unpurified) can be extracted from 1 kg of defatted and dried mycelium of M. circinelloides IBT-83. Pre-dehydration of the mycelium by freeze-drying positively affects both the lipid extraction efficiency and the subsequent recovery of chitosan. The method developed in this study represents a promising approach for increased waste valorisation and supports the production of an industrially important biological product.

Mucorales fungi cause life-threatening mucormycosis in patients with clinical risk factors, such as immunodeficiency. Moreover, they are resistant to several antifungal drugs, highlighting the urgent need for novel therapies. Traditional mammalian models are expensive and raise ethical concerns, thereby limiting their suitability for large-scale studies. We established a silkworm (Bombyx mori) infection model to investigate the pathogenicity of Mucorales and evaluated its relevance to human infection. Strains of Rhizopus arrhizus, Mucor circinelloides, and Cunninghamella bertholletiae induced fatal infections in the silkworm. Grocott-stained silkworm tissue sections revealed hyphal invasion patterns closely resembling those observed in human mucormycosis. In addition, experimental simulation of clinical risk factors (steroid use and iron overload) significantly reduced the median lethal dose (LD50). Treatment with the antifungal drug isavuconazonium prolonged the survival of R. arrhizus-infected silkworms, suggesting the potential use of this model for novel antifungal screening. LD50 estimation for four R. arrhizus strains revealed up to a 100-fold difference in pathogenicity to the silkworm among the tested strains, corresponding to variations in cell surface characteristics. Interestingly, the presence of high-molecular weight (50–100 kDa) cell surface proteins was associated with high pathogenicity among R. arrhizus strains. In conclusion, the silkworm is a viable, human-relevant alternative model to investigate Mucorales infections, with potential for large-scale antimucormycosis drug screening.

This study investigates the diversity and provides a preliminary evaluation of the enzymatic potential of culturable fungi present in two-phase olive mill waste (TPOMW), a lignocellulose- and phenolic-rich agro-industrial by-product generated in large quantities in olive oil-producing countries. Ninety-four isolates, representing 31 species of the phyla Ascomycota, Basidiomycota, and Mucoromycota, were obtained and identified by using ITS, 28S, tef1-α, tub2, rpb2, act, and/or cal sequences. Among the identified taxa, two new Trichoderma species within the Harzianum clade, namely Trichoderma amurcicola (phylogenetically related to T. simile and T. guizhouense) and Trichoderma olivarum (phylogenetically related to T. simmonsii), were described following a multilocus phylogenetic analysis combined with a study of their morphoanatomical features. A rather high phylogenetic divergence was detected in Candida boidinii, Pleurostoma richardsiae, and Mucor circinelloides, while Cladosporium limoniforme, Mucor pseudolusitanicus, Stagonosporopsis ailanthicola, and Talaromyces nanjingensis were recorded for the first time in TPOMW. A preliminary screening revealed 29 species with cellulolytic and/or xylanolytic activities; 26 species displayed dye decolorization capacity, while ligninolytic and laccase activities were restricted to a few taxa. The most promising degraders of lignocellulosics included strains of Cladosporium limoniforme, C. ramotenellum, Fuscoporia ferrea, Peniophora lycii, and Pseudophlebia setulosa. Fungi detected in TPOMW are promising biotechnological tools to be exploited in the frame of circular economy applications.

Fatty acid synthase (FAS) is one of the most important enzymes in lipid biosynthesis, which can catalyze the reaction of acetyl-CoA and malonyl-CoA to produce fatty acids. However, the structure, function, and molecular mechanism of FAS regulating lipid synthesis in the fungus Mucor circinelloides are unclear. In the present study, two encoding fas genes in the high lipid-producing strain WJ11 and low lipid-producing strain CBS277.49 from M. circinelloides based on their genome database were identified and annotated respectively. Bioinformatic analysis confirmed the presence of typical conserved domains in FAS proteins. Phylogenetic analysis revealed the evolutionary relationship of these FAS proteins, and the FAS proteins from WJ11 have similar properties as their counterparts in CBS277.49. Furthermore, transcriptional profiling displayed marked differences in the expression of these fas genes, and fas1 was analyzed to predict the possible functions in lipid metabolism in the high lipid-producing strain WJ11. This is the first report on the structures and functions of FAS proteins in M. circinelloides. This research has suggested the association of fas genes with lipid metabolism at the transcriptional level and contributed to the selection of candidates related to lipid metabolism for further study.

Fungi are pivotal in transitioning to a bio-based, circular economy due to their ability to transform organic material into valuable products such as organic acids, enzymes, and drugs. Mucor circinelloides is a model organism for studying lipogenesis and is particularly promising for its metabolic capabilities in producing oils like TAGs and carotenoids, influenced by environmental factors such as nutrient availability. Notably, strains VI04473 and FRR5020 have been identified for their potential in producing single-cell oils and carotenoids, respectively. Calcium starvation has previously been shown to have strain-specific effects, with VI04473 accumulating more lipids and FRR5020 producing more carotenoids. Here, we used genome sequencing, comparative genomics, transcriptomics, and metabolite phenotyping to explore the genetic basis of lipid and carotenoid production under calcium starvation in these strains. We found extensive genomic rearrangements between these strains, as well as low conservation of gene regulatory responses to calcium depletion. This lack of conservation also applies to genes involved in lipid and carotenoid production, ie the lipidome. Crucially, we identified several metabolic pathways with distinct transcriptional responses to calcium depletion, suggesting the existence of a previously unrecognized, strain-dependent mechanism by which calcium signaling modulates metabolite production. This points to a potentially novel regulatory pathway linking calcium homeostasis to secondary metabolism in fungi, which may be linked to the complex gene family evolution of several lipidome-genes. Our study sheds light on the complexity of the evolution of metabolic networks in M. circinelloides. Understanding these genetic underpinnings can optimize the industrial use of M. circinelloides, enhancing lipid productivity and stress tolerance, and tailoring metabolic profiles for specific applications.

The effect of exogenous addition of metformin hydrochloride on lipid synthesis in Mucor circinelloides WJ11.

Introduction: Mucormycosis is a rare fungal infection caused by ubiquitous fungi belonging to the order Mucorales. It’s most frequently observed in immunocompromised patients or immunocompetent following a significant trauma. We present the case of an immunocompetent woman who developed Mucormycosis after a minor skin trauma.Clinical Decours: A 61-year-old woman, coping with depression and alcohol abuse, was admitted to the hospital due to septic shock caused by a soft tissue infection in her left leg, which developed after a minor skin trauma. Broad-spectrum antibiotic therapy was initiated and subsequently tailored based on specific bacteria identified in soft tissue and blood samples. Due to a worsening wound condition, surgical debridement, along with post-surgical VAC therapy, was performed. Mucor circinelloides was identified through histopathological analysis and cultures of surgical samples. To prevent the risk of a disseminated infection (mortality 70-100%) limb amputation was considered to achieve source control. However, due to the frailty of the patient, we opted for an attempt of conservative approach: bone samples were obtained and tested negative for fungal presence, finally addressing for a potentially avoidable amputation. The patient was successfully treated with surgical curettages, in combination with amphotericin B and isavuconazole.Conclusions: Although rare and more often seen in predisposed patients, Mucormycosis should also be considered in the differential diagnosis of soft tissue infections arising from small skin traumas in immunocompetent patients.

Epimutations modify gene expression and lead to phenotypic variation while the encoding DNA sequence remains unchanged. Epimutations mediated by RNA interference (RNAi) and/or chromatin modifications can confer antifungal drug resistance and may impact virulence traits in fungi. However, whether these epigenetic modifications can be transmitted across generations following sexual reproduction was unclear. This study demonstrates that RNAi epimutations conferring antifungal drug resistance are transgenerationally inherited in the human fungal pathogen Mucor circinelloides. Our research reveals that RNAi-based antifungal resistance follows a DNA sequence-independent, non-Mendelian inheritance pattern. Small RNAs (sRNAs) are the exclusive determinants of inheritance, transmitting drug resistance independently of other known repressive epigenetic modifications. Unique sRNA signature patterns can be traced through inheritance from parent to progeny, further supporting RNA as an alternative molecule for transmitting information across generations. Understanding how epimutations occur, propagate, and confer resistance may enable their detection in other eukaryotic pathogens, provide solutions for challenges posed by rising antimicrobial drug resistance, and advance research on phenotypic adaptability and its evolutionary implications.

Malnutrition in underdeveloped regions is exacerbated by the lack of accessible, nutrient-dense foods and energy-intensive agricultural practices. This study introduces a sustainable non-sterile cultivation method for Pleurotus sapidus (an oyster mushroom species) using fresh Pennisetum giganteum (Giant Juncao grass), a fast-growing C4 grass cultivated on marginal soils. Unlike conventional sterilization-dependent approaches, our method employs lime-saturated water (LW, 4 mL·g−1) to pretreat fresh grass, eliminating the need for drying; a critical advantage in humid regions where biomass degradation occurs rapidly. Coupled with optimized substrate composition (2% CaO, 0.5% corn flour), this strategy achieved robust mycelial growth (0.53 cm·d−1) and high biological efficiency (112.78%). LW treatment altered substrate metabolites and reduced soluble nutrients. L-malic acid and soluble sugars promoted P. sapidus growth, whereas amino acids, available nitrogen (N), phosphorus (P) and potassium (K), and lactic acid inhibited its growth. The Mucor circinelloides was identified as a key contaminant. This scalable strategy transforms underutilized lignocellulosic biomass into nutrient-rich food, aligning with SDG 2 (Zero Hunger) and SDG 12 (Responsible Consumption and Production).

The global market size of animal parasiticides was valued at USD 12.9 billion in 2024. Animal deworming only results in temporary cures with little to no preventive effects; therefore, a strategy that combines animal deworming with prevention is essential in improving the control of helminths. The effectiveness of co-administrating curative and preventive agents and their compatibility were considered based on the parasitophagous fungus Mucor circinelloides, which was developed in edible agar-agar (red seaweed)-carrying dewormers. Accordingly, Petri dishes were prepared with either a biopolymer alone (control, G-C) or with the anthelmintic piperazine (550, 1102, 2210, and 5500 mg/plate) or levamisole (37.5, 75, 150, and 300 mg/plate) and were used to culture the fungus Mucor circinelloides. Strong fungal growth and high numbers of spores were observed in the presence of the anthelmintics. No differences were measured between the control plates and those containing parasiticide drugs. Similar mycelial growth patterns and sporogenesis rates were recorded for different amounts of each anthelmintic. In conclusion, this novel formulation based on biopolymers containing anthelmintics and enriched with the parasitophagous fungus represents a highly promising tool to consider for jointly deworming animals and minimizing the risks of helminth infection. Further studies are in progress to confirm these in vitro results.

Antimicrobial resistance is a global health threat emerging through microbe adaptation, driven by genetic variation, genome plasticity or epigenetic regulation. This study investigates how the Mucor circinelloides species complex adapts to the antifungal natural product FK506. In Mucor bainieri, most resistant strains exhibit unstable phenotypes without genetic changes. Approximately ~50% of FK506-resistant isolates acquire resistance via RNAi-dependent epimutation, where small interfering RNAs (siRNAs) silence fkbA transcription. The remaining isolates undergo heterochromatin-mediated silencing via H3K9 methylation and siRNAs spreading, repressing fkbA and neighboring genes. One isolate retained only heterochromatin marks without detectable siRNAs. A similar mechanism operates in Mucor atramentarius, where FK506 resistance is mediated by ectopic heterochromatin associated with siRNA. Strikingly, heterochromatin-based epimutation inheritance remains stable following in vivo infection. These findings reveal that antifungal resistance can arise through distinct, heritable epigenetic pathways involving RNAi, heterochromatin, or both highlighting adaptive strategies employed by ubiquitous eukaryotic microbial pathogens infecting humans.

ABSTRACT Mucormycosis is an emerging infection caused by pathogenic filamentous fungal species belonging to the Order Mucorales. Mortality associated with mucormycosis is significantly high in patients with compromised immunity. As cell wall is the first fungal component to interact with the host immune system, we characterized cell wall organization and composition of the three most prevalent pathogenic species of Mucorales, Rhizopus arrhizus, Mucor circinelloides, and Lichtheimia corymbifera and studied their immunomodulatory potential. Staining and lectin-/immunolabeling indicated that the spores and germ-tubes of these three Mucorales species have surface-exposed mannans, while germ-tubes showed distinctly distributed β-1,3-glucan. Gas chromatography analysis of the cell wall indicated that glucose polymer is the major fibrillar polysaccharide present in the three species, whereas amorphous components were species-dependent. Specific enzymatic digestion followed by chromatography analysis indicated that β-1,3-glucan, β-1,6-glucan, and amylase digestible glucan constitute firbrillar polysaccharides. Stimulation of human peripheral blood mononuclear cells or THP-1 (human leukemic monocytic) cells with spores or extracted cell wall polysaccharides resulted in the release of pro-inflammatory cytokines regardless of the Mucorales species. Together, the Mucorales species analyzed in this study show a common and species-specific cell wall composition. The cell wall polysaccharides are highly pro-inflammatory, suggesting that undue or excessive inflammation may contribute to the immunopathology of mucormycosis.