Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 μM to >7.2 μM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors

The GPCR antagonist PPTN synergizes with caspofungin providing increased fungicidal activity against Aspergillus fumigatus

ABSTRACT Fungal pathogens pose a serious threat to human health, with Candida and Aspergillus spp. representing some of the most significant opportunistic invaders. Aspergillus fumigatus causes aspergillosis, one of the most prevalent fungal diseases of humans. There is a limited number of drugs available to combat these infections, and antifungal drug resistance is on the rise. In this manuscript, we show 4-[4-(4-Piperidinyl) phenyl]-7-[4-(-(trifluoromethyl) phenyl]-2-naphthalenecarboxylic acid (PPTN), a highly specific antagonist of the human P2Y14 receptor, is a promising antifungal adjuvant against diverse fungal pathogens. PPTN interacts with caspofungin (CAS), ibrexafungerp, voriconazole (VOR), and amphotericin against A. fumigatus CAS- and VOR-resistant clinical isolates, and also CAS against Candida spp and Cryptococcus neoformans. The combination of PPTN and CAS increases cell death in A. fumigatus. In the model yeast Saccharomyces cerevisiae, heterozygous deletion of genes involved in chromatin remodeling results in PPTN hypersensitivity, and in A. fumigatus, PPTN can have increased fungicidal activity when combined with the histone deacetylase inhibitor trichostatin A and the DNA methyltransferase inhibitor 5-azacytidine. Finally, PPTN has reduced toxicity to human immortalized cell lineages and partially clears A. fumigatus conidia infection in A549 pulmonary epithelial cells. Our results indicate that PPTN is a novel adjuvant antifungal drug against fungal diseases caused by A. fumigatus and Candida spp.IMPORTANCEInvasive fungal infections have a high mortality rate, causing more deaths annually than tuberculosis or malaria. Aspergillus fumigatus is the main etiological agent of aspergillosis, one of the most prevalent and deadly fungal diseases. There are few therapeutic options for treating this disease, and treatment commonly fails due to host complications or the emergence of antifungal resistance. Drug repurposing, where existing drugs are deployed for other clinical indications, has increasingly been used in the process of drug discovery. Here, we show that 4-[4-(4-Piperidinyl) phenyl]-7-[4-(-(trifluoromethyl) phenyl]-2-naphthalenecarboxylic acid (PPTN), a highly specific antagonist of the human P2Y14 receptor, when combined with caspofungin (CAS), ibrexafungerp, voriconazole (VOR), and amphotericin can increase the fungicidal activity against not only A. fumigatus CAS- and VOR-resistant clinical isolates but also CAS against Candida spp

Evolutionary origin and population diversity of a cryptic hybrid pathogen

Cryptic fungal pathogens pose disease management challenges due to their morphological resemblance to known pathogens. Here, we investigated the genomes and phenotypes of 53 globally distributed isolates of Aspergillus section Nidulantes fungi and found 30 clinical isolates—including four isolated from COVID-19 patients—were A. latus, a cryptic pathogen that originated via allodiploid hybridization. Notably, all A. latus isolates were misidentified. A. latus hybrids likely originated via a single hybridization event during the Miocene and harbor substantial genetic diversity. Transcriptome profiling of a clinical isolate revealed that both parental subgenomes are actively expressed and respond to environmental stimuli. Characterizing infection-relevant traits—such as drug resistance and growth under oxidative stress—revealed distinct phenotypic profiles among A. latus hybrids compared to parental and closely related species. Moreover, we identified four features that could aid A. latus taxonomic identification. Together, these findings deepen our understanding of the origin of cryptic pathogens.National Science Foundation (NSF) DEB-2110404National Institute of Allergy and Infectious Diseases (NIAID) R01 AI153356Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2016/07870-9, 2021/04977-5Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 301058/2019-9, 404735/2018-5European Union (UE). 847507Ministerio de Ciencia, Innovación y Universidades (MICINN) PID2021-128001OB-I00Agencia Estatal de Investigación (AEI) TED2021-129601B-I0

Aspergillus fumigatus conidial surface-associated proteome reveals factors for fungal evasion and host immunity modulation

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1β, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.</p