Paramyxovirus membrane fusion requires an attachment protein for receptor binding and a fusion protein for membrane fusion triggering. Nipah virus (NiV) attachment protein (G) binds to ephrinB2 or -B3 receptors, and fusion protein (F) mediates membrane fusion. NiV-F is a class I fusion protein and is activated by endosomal cleavage. The crystal structure of a soluble GCN4-decorated NiV-F shows a hexamer-of-trimer assembly. Here, we used single-molecule localization microscopy to quantify the NiV-F distribution and organization on cell and virus-like particle membranes at a nanometer precision. We found that NiV-F on biological membranes forms distinctive clusters that are independent of endosomal cleavage or expression levels. The sequestration of NiV-F into dense clusters favors membrane fusion triggering. The nano-distribution and organization of NiV-F are susceptible to mutations at the hexamer-of-trimer interface, and the putative oligomerization motif on the transmembrane domain. We also show that NiV-F nanoclusters are maintained by NiV-F–AP-2 interactions and the clathrin coat assembly. We propose that the organization of NiV-F into nanoclusters facilitates membrane fusion triggering by a mixed population of NiV-F molecules with varied degrees of cleavage and opportunities for interacting with the NiV-G/receptor complex. These observations provide insights into the in situ organization and activation mechanisms of the NiV fusion machinery.

Non-inheritable antibiotic or phenotypic resistance ensures bacterial survival during antibiotic treatment. However, exogenous factors promoting phenotypic resistance are poorly defined. Here, we demonstrate that Vibrio alginolyticus are recalcitrant to killing by a broad spectrum of antibiotics under high magnesium. Functional metabolomics demonstrated that magnesium modulates fatty acid biosynthesis by increasing saturated fatty acid biosynthesis while decreasing unsaturated fatty acid production. Exogenous supplementation of unsaturated and saturated fatty acids increased and decreased bacterial susceptibility to antibiotics, respectively, confirming the role of fatty acids in antibiotic resistance. Functional lipidomics revealed that glycerophospholipid metabolism is the major metabolic pathway remodeled by magnesium, where phosphatidylethanolamine biosynthesis is reduced and phosphatidylglycerol production is increased. This process alters membrane composition, increasing membrane polarization, and decreasing permeability and fluidity, thereby reducing antibiotic uptake by V. alginolyticus. These findings suggest the presence of a previously unrecognized metabolic mechanism by which bacteria escape antibiotic killing through the use of an environmental factor.