A series of studies was conducted to test the antifungal activity of clavicipitaceous endophytes and to identify potential fungal inhibitors in this symbiotic infection. A diverse group of endophytes was screened for antifungal activity using organic extracts from liquid fermentation cultures. Fungal inhibitors were purified from fermentation cultures of Epichloë festucae using a bioassay-directed extraction with Cryphonectria parasitica as the test organism. Compounds shown to have antifungal activity were subsequently identified using NMR and GC-MS. Extracts from a wide range of fungal isolates had various degrees of antifungal activity, but the greatest antifungal activity was observed in E. festucae and Neotyphodium tembladerae. Three types of inhibitors were isolated from a batch culture of E. festucae, including several indole derivatives, a sesquiterpene, and a diacetamide. Among the indole derivatives, indole-3-acetic acid and indole-3-ethanol were identified as the major indoles. These compounds were previously reported in endophytic fungi, and this study suggests a role in host disease resistance against other pathogens. The diversity in fungal inhibitors produced by this endophyte also suggests that fungal inhibitors may act additively or synergistically to reduce colonization of endophyte-infected hosts by potential fungal competitors.
A phage library displaying 10
10
variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD and the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.
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