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

Abstract

Lysobacter are new biocontrol agents known for their prolific production of lytic enzymes and bioactive metabolites. L. enzymogenes 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 L. enzymogenes interacts with fungal prey is not well understood. Here, we found that the production of HSAF and analogs in L. enzymogenes OH11 was significantly induced in media supplemented with ground fungal mycelia or chitin. In the OH11 genome, we identified a gene (LeLPMO10A) that was annotated to encode a chitin-binding protein. The stimulation of HSAF and analogs by chitin was diminished when LeLPMO10A was deleted. We expressed the gene in E. coli and demonstrated that purified LeLPMO10A oxidatively cleaved chitin into oligomeric products, including 1,5 δ-lactones and aldonic acids. The results revealed that LeLPMO10A encodes a lytic polysaccharide monooxygenase, which has not been reported in Lysobacter. 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 in vitro 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 LeLPMO10A was deleted. Together, the study revealed a previously uncharacterized synergistic strategy utilized by the predatory Lysobacter during interaction with fungal prey.

On the expansion of biological functions of lytic polysaccharide monooxygenases

“…Literature analysis of the occurrence of lytic polysaccharide monooxygenases (LPMOs) found in various pathosystems. (a, b) Number of research articles reporting an LPMO (or LPMO‐like) shown to be relevant by either transcriptomic, proteomic and/or enzyme characterisation in different emitters (Kawada et al ., 2008; Chaudhuri et al ., 2010; Valente et al ., 2011; Stauder et al ., 2012; Wong et al ., 2012; Yakovlev et al ., 2012; Garcia‐Gonzalez et al ., 2014; Loose et al ., 2014; Chiu et al ., 2015; Hamre et al ., 2015; Huang et al ., 2015; Paspaliari et al ., 2015; Zhang et al ., 2015; Mekasha et al ., 2016; Shukla et al ., 2016; Liu et al ., 2017, 2018, 2020; Hegnar et al ., 2019; Yadav et al ., 2019; Garcia‐Santamarina et al ., 2020; Labourel et al ., 2020; Li et al ., 2020; Munzone et al ., 2020; Askarian et al ., 2021; Polonio et al ., 2021; Sabbadin et al ., 2021a,b; Yue et al ., 2021), and displayed as (a) a function of the year of publication per emitter and (b) an emitter–target map, in which the circle size indicates the number of articles and the colour indicates the protein family. Note that the ‘preferred target’ shows the potential target of the emitter, and not necessarily the target of the LPMO, which in several cases remains to be fully demonstrated (see main text).…”

“…Studies on LPMOs from bacteria such as Vibrio cholerae , Paenibacillus larvae , Listeria monocytogenes , Pseudomonas aeruginosa and Lysobacter enzymogenes have shown this class of enzymes to be involved in pathogenesis and virulence roles (Garcia‐Gonzalez et al ., 2014; Loose et al ., 2014; Paspaliari et al ., 2015; Askarian et al ., 2021; Yue et al ., 2021). As virulence factors, LPMOs may be studied as potential targets for synthesis of inhibitors against the pathogen.…”

“…Lysobacter enzymogenes is an interesting bacterium with the ability to produce bioactive metabolites against a wide spectrum of fungal pathogens of plants and humans. A chitinolytic AA10 LPMO secreted by L. enzymogenes ( Le LPMO10A) was seen to induce the production of antimicrobial compounds called ‘heat stable antifungal factor’ and its analogues (Yue et al ., 2021). The authors further suggested a mechanism of action, yet to be fully demonstrated, for Le LPMO10A: the LPMO would cleave the recalcitrant chitin present in the fungal pathogen cell wall, enabling thereby the delivery of the antifungal factors via outer membrane vesicles.…”

Lysobacter terrestris sp. nov., isolated from soil

“… Lysobacter colonies are mucoid and cream, pink or yellow-brown in colour. Most Lysobacter members can be distinguished from closely related microbes due to their genomic high G+C content (ranging between 65–72 %), lytic activity against other bacteria, fungi, algae and nematodes [4–7], and the absence of flagella excepts some strains ( Lysobacter xanthus and Lysobacter helvus ) [8, 9]. The genus Lysobacter comprises 62 published species (https://lpsn.dsmz.de/genus/lysobacter), including Lysobacter terrae [10], Lysobacter niabensis [11], Lysobacter oryzae [12], Lysobacter yangpyeongensis [13] and Lysobacter enzymogenes [4].…”

Antifungal weapons of Lysobacter, a mighty biocontrol agent

“…These vesicles bud off from the outer membranes of Gram‐negative bacteria and may contain various cellular components including DNA, RNA, proteins and small molecules (Schwechheimer and Kuehn, 2015). The Lysobacter OMVs were shown to contain not only HSAF but also other antifungal enzymes, like lytic polysaccharide monooxygenase (Yue et al ., 2021). The role of OMVs in antifungal toxin delivery in Lysobacter clearly requires further investigation.…”