Outer Membrane Vesicle-Mediated Codelivery of the Antifungal HSAF Metabolites and Lytic Polysaccharide Monooxygenase in the Predatory <i>Lysobacter enzymogenes</i>

Abstract

<i>Lysobacter</i> are new biocontrol agents known for their prolific production of lytic enzymes and bioactive metabolites. <i>L. enzymogenes</i> is a predator of fungi and produces several structurally distinct antimicrobial compounds, such as the antifungal HSAF (heat stable antifungal factor) and analogs. The mechanism by which <i>L. enzymogenes</i> interacts with fungal prey is not well understood. Here, we found that the production of HSAF and analogs in <i>L. enzymogenes</i> OH11 was significantly induced in media supplemented with ground fungal mycelia or chitin. In the OH11 genome, we identified a gene (<i>LeLPMO10A</i>) that was annotated to encode a chitin-binding protein. The stimulation of HSAF and analogs by chitin was diminished when <i>LeLPMO10A</i> was deleted. We expressed the gene in <i>E. coli</i> and demonstrated that purified LeLPMO10A oxidatively cleaved chitin into oligomeric products, including 1,5 δ-lactones and aldonic acids. The results revealed that <i>LeLPMO10A</i> encodes a lytic polysaccharide monooxygenase, which has not been reported in <i>Lysobacter</i>. The metabolite analysis, antifungal assay, and proteomic analysis showed that the antifungal compounds and the chitin-cleaving LeLPMO10A are colocalized in outer membrane vesicles. The enzymatic products that resulted from <i>in vitro</i> LeLPMO10A-cleaved chitin also significantly induced HSAF and analogs in OH11. Scanning electron microscopic analysis indicated that spherical vesicles were formed outside of OH11 cells, and fewer OH11 cells were observed to attach to fungal hyphae when <i>LeLPMO10A</i> was deleted. Together, the study revealed a previously uncharacterized synergistic strategy utilized by the predatory <i>Lysobacter</i> during interaction with fungal prey.