Lipopeptides from Bacillus velezensis ZLP-101 and their mode of action against bean aphids Acyrthosiphon pisum Harris.

BACKGROUND: Natural products are important sources for the discovery of new biopesticides to control the worldwide destructive pests Acyrthosiphon pisum Harris. Here, insecticidal substances were discovered and characterized from the secondary metabolites of the bio-control microorganism Bacillus velezensis strain ZLP-101, as informed by whole-genome sequencing and analysis. RESULTS: The genome was annotated, revealing the presence of four potentially novel gene clusters and eight known secondary metabolite synthetic gene clusters. Crude extracts, prepared through ammonium sulfate precipitation, were used to evaluate the effects of strain ZLP-101 on Acyrthosiphon pisum Harris aphid pests via exposure experiments. The half lethal concentration (LC50) of the crude extract from strain ZLP-101 against aphids was 411.535 mg/L. Preliminary exploration of the insecticidal mechanism revealed that the crude extract affected aphids to a greater extent through gastric poisoning than through contact. Further, the extracts affected enzymatic activities, causing holes to form in internal organs along with deformation, such that normal physiological activities could not be maintained, eventually leading to death. Isolation and purification of extracellular secondary metabolites were conducted in combination with mass spectrometry analysis to further identify the insecticidal components of the crude extracts. A total of 15 insecticidal active compounds were identified including iturins, fengycins, surfactins, and spergualins. Further insecticidal experimentation revealed that surfactin, iturin, and fengycin all exhibited certain aphidicidal activities, and the three exerted synergistic lethal effects. CONCLUSIONS: This study improved the available genomic resources for B. velezensis and serves as a foundation for comprehensive studies of the insecticidal mechanism by Bacillus velezensis ZLP-101 in addition to the active components within biological control strains.

Modifying the Catalytic Activity of Lipopeptide Assemblies with Nucleobases.

Biohybrid catalysts that operate in aqueous media are intriguing for systems chemistry. In this paper, we investigate whether control over the self-assembly of biohybrid catalysts can tune their properties. As a model, we use the catalytic activity of functional hybrid molecules consisting of a catalytic H-dPro-Pro-Glu tripeptide, derivatized with fatty acid and nucleobase moieties. This combination of simple biological components merged the catalytic properties of the peptide with the self-assembly of the lipid, and the structural ordering of the nucleobases. The biomolecule hybrids self-assemble in aqueous media into fibrillar assemblies and catalyze the reaction between butanal and nitrostyrene. The interactions between the nucleobases enhanced the order of the supramolecular structures and affected their catalytic activity and stereoselectivity. The results point to the significant control and ordering that nucleobases can provide in the self-assembly of biologically inspired supramolecular catalysts.

Engagement with the TCR induces plasticity in antigenic ligands bound to MHC class I and CD1 molecules.

Complementarity-determining regions (CDRs) of αß T-cell receptors (TCRs) sense peptide-bound MHC (pMHC) complexes via chemical interactions, thereby mediating antigen specificity and MHC restriction. Flexible finger-like movement of CDR loops contributes to the establishment of optimal interactions with pMHCs. In contrast, peptide ligands captured in MHC molecules are considered more static because of the rigid hydrogen-bond network that stabilizes peptide ligands in the antigen-binding groove of MHC molecules. An array of crystal structures delineating pMHC complexes in TCR-docked and TCR-undocked forms is now available, which enables us to assess TCR engagement-induced conformational changes in peptide ligands. In this short review, we overview conformational changes in MHC class I-bound peptide ligands upon TCR docking, followed by those for CD1-bound glycolipid ligands. Finally, we analyze the co-crystal structure of the TCR:lipopeptide-bound MHC class I complex that we recently reported. We argue that TCR engagement-induced conformational changes markedly occur in lipopeptide ligands, which are essential for exposure of a primary T-cell epitope to TCRs. These conformational changes are affected by amino acid residues, such as glycine, that do not interact directly with TCRs. Thus, ligand recognition by specific TCRs involves not only T-cell epitopes but also non-epitopic amino acid residues. In light of their critical function, we propose to refer to these residues as non-epitopic residues affecting ligand plasticity and antigenicity (NR-PA).

Total synthesis of antifungal lipopeptide iturin A analogues and evaluation of their bioactivity against F. graminearum.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the ß-amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure-activity relationship studies. The assays also highlight the importance of the ß-amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

Lipopeptides from Bacillus velezensis induced apoptosis-like cell death in the pathogenic fungus Fusarium concentricum.

AIMS: Stem rot caused by Fusarium concentricum is a new disease of Paris polyphylla reported by our research group. The present study investigates the growth inhibitory and apoptotic effects of Bacillus velezensis FJAT-54560 lipopeptide against F. concentricum. METHODS AND RESULTS: HPLC preparation and LC-MS analysis results show that the crude lipopeptides secreted by Bacillus velezensis FJAT-54560 isolated from Jasminum sambac consist of C14-17 iturin A, C14 fengycin B, C16 fengycin A/A2, C18 fengycin A, C20 fengycin B2, C21 fengycin A2, C22-23 fengycin A, C12-16 surfactin A, and C15 surfactin A derivatives. The mass ratios (g/g) of iturin, fengycin, and surfactin in lipopeptides are 2.40, 67.51, and 30.08%, respectively. Through inhibition zone and inhibition rate experiments, we found that crude lipopeptides and purified fengycin exhibit strong antifungal activity against F. concentricum, including accumulation of reactive oxygen species, loss of mitochondrial membrane potential, DNA fragmentation, Ca2+ accumulation, chromatin condensation, and phosphatidylserine externalization. Transcriptomic analysis indicates that crude lipopeptide-induced apoptosis in F. concentricum cells may be mediated by apoptosis-inducing factors and apoptosis mediators and can serve as a metacaspase-independent model. CONCLUSION: Lipopeptides from Bacillus velezensis FJAT-54560 can control the pathogenic fungus F. concentricum by inducing apoptosis.

Homo and Hetero-Branched Lipopeptide Dendrimers: Synthesis and Antimicrobial Activity.

Di-branched and tetra-branched versions of a previously reported analogue of the lipopeptide battacin were successfully synthesised using thiol-maleimide click and 1, 2, 3-triazole click chemistry. Antimicrobial studies against drug resistant clinical isolates of Escherichia coli (ESBL E. coli Ctx-M14), Pseudomonas aeruginosa (P. aeruginosa Q502), and Methicillin resistant Staphylococcus aureus (MRSA ATCC 33593), as well as clinically isolated Acinetobacter baumannii (A. baumannii ATCC 19606), and P. aeruginosa (ATCC 27853), revealed that the dendrimeric peptides have antimicrobial activity in the low micromolar range (0.5 -- 4 µM) which was 10 times more potent than the monomer peptides. Under high salt concentrations (150 mM NaCl, 2 mM MgCl2, and 2.5 mM CaCl2) the di-branched lipopeptides retained their antimicrobial activity while the monomer peptides were not active (>100 µM). The di-branched triazole click lipopeptide, Peptide 12, was membrane lytic, showed faster killing kinetics, and exhibited antibiofilm activity against A. baumannii and MRSA and eradicated > 85 % preformed biofilms at low micromolar concentrations. The di-branched analogues were > 30-fold potent than the monomers against Candida albicans. Peptide 12 was not haemolytic (HC10 = 932.12 µM) and showed up to 40-fold higher selectivity against bacteria and fungi than the monomer peptide. Peptide 12 exhibited strong proteolytic stability (>80 % not degraded) in rat serum over 24 h whereas > 95 % of the thiol-maleimide analogue (Peptide 10) was degraded. The tetra-branched peptides showed comparable antibacterial potency to the di-branched analogues. These findings indicate that dual branching using triazole click chemistry is a promising strategy to improve the antimicrobial activity and proteolytic stability of battacin based lipopeptides. The information gathered can be used to build effective antimicrobial dendrimeric peptides as new peptide antibiotics.

Inhibition mechanism of crude lipopeptide from Bacillus subtilis against Aeromonas veronii growth, biofilm formation, and spoilage of channel catfish flesh.

Aeromonas veronii is associated with food spoilage and some human diseases, such as diarrhea, gastroenteritis, hemorrhagic septicemia or asymptomatic and even death. This research investigated the mechanism of the growth, biofilm formation, virulence, stress resistance, and spoilage potential of Bacillus subtilis lipopeptide against Aeromonas veronii. Lipopeptides suppressed the transmembrane transport of Aeromonas veronii by changing the cell membrane's permeability, the structure of membrane proteins, and Na+/K+-ATPase. Lipopeptide significantly reduced the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) by 86.03% and 56.12%, respectively, ultimately slowing Aeromonas veronii growth. Lipopeptides also restrained biofilm formation by inhibiting Aeromonas veronii motivation and extracellular polysaccharide secretion. Lipopeptides downregulated gene transcriptional levels related to the virulence and stress tolerance of Aeromonas veronii. Furthermore, lipopeptides treatment resulted in a considerable decrease in the extracellular protease activity of Aeromonas veronii, which restrained the decomposing of channel catfish flesh. This research provides new insights into lipopeptides for controlling Aeromonas veronii and improving food safety.

Lipopeptide-mediated bacterial interaction enables cooperative predator defense.

Bacteria are inherently social organisms whose actions should ideally be studied within an interactive ecological context. We show that the exchange and modification of natural products enables two unrelated bacteria to defend themselves against a common predator. Amoebal predation is a major cause of death in soil bacteria and thus it exerts a strong selective pressure to evolve defensive strategies. A systematic analysis of binary combinations of coisolated bacteria revealed strains that were individually susceptible to predation but together killed their predator. This cooperative defense relies on a Pseudomonas species producing syringafactin, a lipopeptide, which induces the production of peptidases in a Paenibacillus strain. These peptidases then degrade the innocuous syringafactin into compounds, which kill the predator. A combination of bioprospecting, coculture experiments, genome modification, and transcriptomics unravel this novel natural product-based defense strategy.

The Physicochemical and Functional Properties of Biosurfactants: A Review.

Surfactants, also known as surface-active agents, have emerged as an important class of compounds with a wide range of applications. However, the use of chemical-derived surfactants must be restricted due to their potential adverse impact on the ecosystem and the health of human and other living organisms. In the past few years, there has been a growing inclination towards natural-derived alternatives, particularly microbial surfactants, as substitutes for synthetic or chemical-based counterparts. Microbial biosurfactants are abundantly found in bacterial species, predominantly Bacillus spp. and Pseudomonas spp. The chemical structures of biosurfactants involve the complexation of lipids with carbohydrates (glycolipoproteins and glycolipids), peptides (lipopeptides), and phosphates (phospholipids). Lipopeptides, in particular, have been the subject of extensive research due to their versatile properties, including emulsifying, antimicrobial, anticancer, and anti-inflammatory properties. This review provides an update on research progress in the classification of surfactants. Furthermore, it explores various bacterial biosurfactants and their functionalities, along with their advantages over synthetic surfactants. Finally, the potential applications of these biosurfactants in many industries and insights into future research directions are discussed.

Production, characterization, and potential applications of lipopeptides in food systems: A comprehensive review.

Lipopeptides are a class of lipid-peptide-conjugated compounds with differing structural features. This structural diversity is responsible for their diverse range of biological properties, including antimicrobial, antioxidant, and anti-inflammatory activities. Lipopeptides have been attracting the attention of food scientists due to their potential as food additives and preservatives. This review provides a comprehensive overview of lipopeptides, their production, structural characteristics, and functional properties. First, the classes, chemical features, structure-activity relationships, and sources of lipopeptides are summarized. Then, the gene expression and biosynthesis of lipopeptides in microbial cell factories and strategies to optimize lipopeptide production are discussed. In addition, the main methods of purification and characterization of lipopeptides have been described. Finally, some biological activities of the lipopeptides, especially those relevant to food systems along with their mechanism of action, are critically examined.

Microbial lipopeptides: their pharmaceutical and biotechnological potential, applications, and way forward.

As lead molecules, cyclic lipopeptides with antibacterial, antifungal, and antiviral properties have garnered a lot of attention in recent years. Because of their potential, cyclic lipopeptides have earned recognition as a significant class of antimicrobial compounds with applications in pharmacology and biotechnology. These lipopeptides, often with biosurfactant properties, are amphiphilic, consisting of a hydrophilic moiety, like a carboxyl group, peptide backbone, or carbohydrates, and a hydrophobic moiety, mostly a fatty acid. Besides, several lipopeptides also have cationic groups that play an important role in biological activities. Antimicrobial lipopeptides can be considered as possible substitutes for antibiotics that are conventional to address the current drug-resistant issues as pharmaceutical industries modify the parent antibiotic molecules to render them more effective against antibiotic-resistant bacteria and fungi, leading to the development of more resistant microbial strains. Bacillus species produce lipopeptides, which are secondary metabolites that are amphiphilic and are typically synthesized by non-ribosomal peptide synthetases (NRPSs). They have been identified as potential biocontrol agents as they exhibit a broad spectrum of antimicrobial activity. A further benefit of lipopeptides is that they can be produced and purified biotechnologically or biochemically in a sustainable manner using readily available, affordable, renewable sources without harming the environment. In this review, we discuss the biochemical and functional characterization of antifungal lipopeptides, as well as their various modes of action, method of production and purification (in brief), and potential applications as novel antibiotic agents.

Co-production of surfactin and fengycin by Bacillus subtilis BBW1542 isolated from marine sediment: a promising biocontrol agent against foodborne pathogens.

Pathogenic bacteria contaminations and related diseases in food industries is an urgent issue to solve. The present study aimed to explore natural food biopreservatives from microorganisms. Using dilution-plate method, a strain BBW1542 with antimicrobial activities against various foodborne pathogenic bacteria was isolated from the seabed silt of Beibu Gulf, which was identified as Bacillus subtilis by the morphological observation and 16S rDNA sequences. The antimicrobial substances of B. subtilis BBW1542 exhibited an excellent stability under cool/heat treatment, UV irradiation, acid/alkali treatment, and protease hydrolysis. The genome sequencing analysis and antiSMASH prediction indicated that B. subtilis BBW1542 contained the gene cluster encoding lipopeptides and bacteriocin subtilosin A. MALDI-TOF-MS analysis showed that the lipopeptides from B. subtilis BBW1542 contained C14 and C15 surfactin homologues, together with fengycin homologues of C18 fengycin A/C16 fengycin B and C19 fengycin A/C17 fengycin B. In silico analysis showed that an eight-gene (sboA-albABCDEFG) operon was involved in the biosynthesis of subtilosin A in B. subtilis BBW1542, and the encoded subtilosin A presented an evident closed-loop structure containing 35 amino acids with a molecular weight of 3425.94 Da. Overall, the antagonistic B. subtilis BBW1542 displayed significant resource value and offered a promising alternative in development of food biopreservation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05864-3.

Molecular characterization and antifungal activity of lipopeptides produced from Bacillus subtilis against plant fungal pathogen Alternaria alternata.

Over 380 host plant species have been known to develop leaf spots as a result of the fungus Alternaria alternata. It is an aspiring pathogen that affects a variety of hosts and causes rots, blights, and leaf spots on different plant sections. In this investigation, the lipopeptides from the B. subtilis strains T3, T4, T5, and T6 were evaluated for their antifungal activities. In the genomic DNA, iturin, surfactin, and fengycin genes were found recovered from B. subtilis bacterium by PCR amplification. From different B. subtilis strains, antifungal Lipopeptides were extracted, identified by HPLC, and quantified with values for T3 (24 g/ml), T4 (32 g/ml), T5 (28 g/ml), and T6 (18 g/ml). To test the antifungal activity, the isolated lipopeptides from the B. subtilis T3, T4, T5, and T6 strains were applied to Alternaria alternata at a concentration of 10 g/ml. Lipopeptides were found to suppress Alternaria alternata at rates of T3 (75.14%), T4 (75.93%), T5 (80.40%), and T6 (85.88%). The T6 strain outperformed the other three by having the highest antifungal activity against Alternaria alternata (85.88%).

A cyclic lipopeptide produced by an antagonistic bacterium relies on its tail and transient receptor potential-type Ca2+ channels to immobilize a green alga.

The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide (CLiP) orfamide A, which triggers a Ca2+ signal causing rapid deflagellation of the microalga Chlamydomonas reinhardtii. We performed chemical synthesis of orfamide A derivatives and used an aequorin reporter line to measure their Ca2+ responses. Immobilization of algae was studied using a modulator and mutants of transient receptor potential (TRP)-type channels. By investigating targeted synthetic orfamide A derivatives, we found that N-terminal amino acids of the linear part and the terminal fatty acid region are important for the specificity of the Ca2+ -signal causing deflagellation. Molecular editing indicates that at least two distinct Ca2+ -signaling pathways are triggered. One is involved in deflagellation (Thr3 change, fatty acid tail shortened by 4C), whereas the other still causes an increase in cytosolic Ca2+ in the algal cells, but does not cause substantial deflagellation (Leu1 change, fatty acid hydroxylation, fatty acid changes by 2C). Using mutants, we define four TRP-type channels that are involved in orfamide A signaling; only one (ADF1) responds additionally to low pH. These results suggest that the linear part of the CLiP plays one major role in Ca2+ signaling, and that orfamide A uses a network of algal TRP-type channels for deflagellation.

Development of truncated Battacin antimicrobials featuring novel N-terminal fatty acids with an excellent safety profile.

Octapeptin B5 peptides containing a novel fatty acids have been found to have enhanced antibacterial activity against Staphylococcus aureus and also have an excellent safety profile. Cyclic lipopeptides such as the polymyxins and battacin are potent antibacterial agents. It has been shown that truncated, non-linear, versions of these agents (e.g. octapeptin B5) can retain the activity of the more complex cyclic compounds. In this work the synthesis of Octapeptin B5 peptides containing a range of novel fatty acids is reported. Many of these lipopeptides have been found to have enhanced antibacterial activity against Staphylococcus aureus compared to Octapeptin B5 whilst also having an excellent safety profile in haemolytic and cytotoxicity assays.

Lipopeptides from an isolate of Bacillus subtilis complex have inhibitory and antibiofilm effects on Fusarium solani.

Bacillus subtilis species complex is known as lipopeptide-producer with biotechnological potential for pharmaceutical developments. This study aimed to identify lipopeptides from a bacterial isolate and evaluate their antifungal effects. Here, we isolated and identified a lipopeptide-producing bacterium as a species of Bacillus subtilis complex (strain UL-1). Twenty lipopeptides (six iturins, six fengycins, and eight surfactins) were identified in the crude extract (CE) and fractions (F1, F2, F3, and F4), and the highest content of total lipopeptides was observed in CE and F2. The chemical quantification data corroborate with the hemolytic and antifungal activities that CE and F2 were the most hemolytic and inhibited the fungal growth at lower concentrations against Fusarium spp. In addition, they caused morphological changes such as shortening and/or atypical branching of hyphae and induction of chlamydospore-like structure formation, especially in Fusarium solani. CE was the most effective in inhibiting the biofilm formation and in disrupting the mature biofilm of F. solani reducing the total biomass and the metabolic activity at concentrations ≥ 2 µg/mL. Moreover, CE significantly inhibited the adherence of F. solani conidia on contact lenses and nails as well as disrupted the pre-formed biofilms on nails. CE at 100 mg/kg was nontoxic on Galleria mellonella larvae, and it reduced the fungal burden in larvae previously infected by F. solani. Taken together, the lipopeptides obtained from strain UL-1 demonstrated a potent anti-Fusarium effect inducing morphological alterations and antibiofilm activities. Our data open further studies for the biotechnological application of these lipopeptides as potential antifungal agents. KEY POINTS: • Lipopeptides inhibit Fusarium growth and induce chlamydospore-like structures. • Lipopeptides hamper the adherence of conidia and biofilms of Fusarium solani. • Iturins, fengycins, and surfactins were associated with antifungal effects.

A novel Bacillus sp. with antagonistic activity against a plant pathogen, Fusarium graminearum, and its potential antagonistic mechanism.

Fusarium head blight (FHB) is a wheat disease caused by the plant pathogen Fusarium graminearum, which leads to crop yield losses and agricultural economic losses, as well as poses a threat to the environment and human health. Effective biocontrol of F. graminearum is urgent. An antagonistic strain HZ-5 with 59.2% antagonistic activity against F. graminearum in vitro had been isolated from sea mud of Haizhou Bay using a dual-culture assay, which was highly homologous with Bacillus halosaccharovorans according to the 16S rRNA sequence. The antagonistic activity of HZ-5 had been further studied. HZ-5 had a broad range of antagonistic activity against another six plant pathogenic fungi and was effective in controlling FHB of wheat in pot experiment. The substances with antagonistic activity were temperature insensitive, and had been purified by HPLC (High Performance Liquid Chromatography) to prove to be secreted lipopeptides. The antagonistic substances induced the biosynthesis of chitin and glycerol, while ergosterol , cholesterol, and phosphatidylcholine reduced their inhibitory effects on F. graminearum. These data would be helpful to provide a better biocontrol strain against FHB, and to provide important basis to elucidate the antagonistic mechanism of biocontrol.

Composition and activity of antifungal lipopeptides produced by Bacillus spp. in daqu fermentation.

Daqu is a solid-state fermentation and saccharification starter for the Chinese liquor baijou. During the daqu stage, amylolytic and proteolytic enzymes are produced by Bacillus and fungi. Bacillus spp. also produce lipopeptides with a broad spectrum of antimicrobial activities but direct evidence for their impact on community assembly in daqu is lacking. This study aimed to study the interaction between Bacillus spp. and fungi in daqu models. The antifungal activity of surfactin, fengycin, and iturin A was initially assessed in vitro. Iturin A displayed the strongest antifungal activity (MIC = 10-50 mg/L). In situ antifungal activity of B. amyloliquefaciens and B. velezensis against molds was observed in a simple daqu model inoculated with single strains of Bacillus species. Formation of lipopeptides in situ was supported by quantification of mRNA encoding for enzymes for surfactin, fengycin, and iturin A biosynthesis. In situ antifungal activity of Bacillus species was also observed in a complex daqu model that was inoculated with 8 bacterial or fungal strains plus one of the three strains of Bacillus. A relationship of lipopeptides to in situ antifungal activity was further supported by detection of the lipopeptides by liquid chromatography coupled to mass spectrometry. Both results indicated that B velezensis FUA2155 had higher antifungal activity in the daqu model, and was the only strain that produced multiple iturin A congeners in situ. Taken together, this study provides evidence that production of lipopeptides by Bacillus species in daqu may impact community assembly and hence product quality.

Recent Advances in Polypeptide Antibiotics Derived from Marine Microorganisms.

In the post-antibiotic era, the rapid development of antibiotic resistance and the shortage of available antibiotics are triggering a new health-care crisis. The discovery of novel and potent antibiotics to extend the antibiotic pipeline is urgent. Small-molecule antimicrobial peptides have a wide variety of antimicrobial spectra and multiple innovative antimicrobial mechanisms due to their rich structural diversity. Consequently, they have become a new research hotspot and are considered to be promising candidates for next-generation antibiotics. Therefore, we have compiled a collection of small-molecule antimicrobial peptides derived from marine microorganisms from the last fifteen years to show the recent advances in this field. We categorize these compounds into three classes-cyclic oligopeptides, cyclic depsipeptides, and cyclic lipopeptides-according to their structural features, and present their sources, structures, and antimicrobial spectrums, with a discussion of the structure activity relationships and mechanisms of action of some compounds.

Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides.

The bacterial pathogen Pseudomonas tolaasii severely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated helper bacteria of the genus Mycetocola (Mycetocola tolaasinivorans and Mycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found that Mycetocola spp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of the Mycetocola proteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.