Identification of peptides from honeybee gut symbionts as potential antimicrobial agents against Melissococcus plutonius

Abstract

Eusocial pollinators are crucial elements in global agriculture. The honeybees and bumblebees are associated with a simple yet host-restricted gut community, which protect the hosts against pathogen infections. Recent genome mining has led to the discovery of biosynthesis pathways of bioactive natural products mediating microbe-microbe interactions from the gut microbiota. Here, we investigate the diversity of biosynthetic gene clusters in the bee gut microbiota by analyzing 477 genomes from cultivated bacteria and metagenome-assembled genomes. We identify 744 biosynthetic gene clusters (BGCs) covering multiple chemical classes. While gene clusters for the post-translationally modified peptides are widely distributed in the bee guts, the distribution of the BGC classes varies significantly in different bee species among geographic locations, which is attributed to the strain-level variation of bee gut members in the chemical repertoire. Interestingly, we find that Gilliamella strains possessing a thiopeptide-like BGC show potent activity against the pathogenic Melissococcus plutonius. The spectrometry-guided genome mining reveals a RiPP-encoding BGC from Gilliamella with a 10 amino acid-long core peptide exhibiting antibacterial potentials. This study illustrates the widespread small-molecule-encoding BGCs in the bee gut symbionts and provides insights into the bacteria-derived natural products as potential antimicrobial agents against pathogenic infections.

Fig. 1. Overview of BGCs predicted in honeybee and bumblebee gut bacteria.

a Overall BGCs were identified from 477 bee gut microbiota genomes. The numbers inside the circle indicate the count of different BGC types. b Whole-genome phylogenetic tree of strains from honeybees and bumblebees with the maximum-likelihood algorithm by PhyloPhlAn 3.0 that uses the 400 universal marker genes. Layers surrounding the genomic trees indicate the classes of BGC, and the heatmap shows the numbers of genes belonging to BGCs in each strain, as predicted by antiSMASH. c A bar graph showing the relative abundance of BGC types in different bacteria genera from honey and bumble bee gut. Source data are provided as a Source Data file.

Fig. 2. BGCs of the gut microbiota distinguish between honey and bumble bee species.

a Network depicting the similarity between putative BGCs identified from bacterial genomes of the bee gut microbiota and a curated collection of functionally characterized BGCs (MIBiG, 2502 sequences). The BGCs were clustered into GCFs and separated into seven classes with BiG-SCAPE. Only GCFs containing at least five BGCs were shown here. Nodes in the network represent BGCs, and the edges connect BGCs with a similarity ≥0.7, defined by BiG-SCAPE. b PCA analysis shows the BGC distribution in the gut of A. cerana, A. mellifera, and B. terrestris individuals. Group differences were tested by one-way permutational multivariate ANOVA (PERMANOVA). c The distribution of RiPPs across bumblebee (B. terrestris) and honeybee (A. cerana, A. mellifera) gut metagenomes. Each column represents one bee gut sample. The relative abundance of BGCs (RPKM) was log-transformed and normalized by the median. Source data are provided as a Source Data file.

Fig. 3. Phylogenetic diversity of lanthipeptide BGCs code by Lactobacillus Firm5.

a Phylogenetic relationships of the Lactobacillus Firm5 coding lanthipeptide BGCs. Cladograms are maximum-likelihood trees inferred by GTDB-tk based on the amino acid sequences of bacterial marker genes. Layers surrounding the genomic trees indicate four classes of lanthipeptides in Lactobacillus Firm5 genomes, as predicted by antiSMASH. A full tree is shown in Supplementary Fig. 1. Gray circles indicate gene presence, and empty circles indicate gene absence. b Syntenic loci of core biosynthetic genes, precursor peptide, additional biosynthetic genes, and transporter genes for lanthipeptide in Lactobacillus Firm5 strains. Homologous genes are connected by gray bars. c Schematic representation of the four classes of lanthipeptide synthetases, highlighting conserved motifs. LanB, lanthipeptide dehydratase; LanC, lanthipeptide cyclase; LanM, class II lanthipeptide synthetase; LanKC, class III synthetase; LanL, class IV lanthipeptide synthetase. The yellow bars in the lyase and kinase domains from LanKC and LanL indicate conserved regions that are important for catalytic activity. d Predicted three precursor peptides dehydration reactions during the LanM of strain ESL0230 deduced from antiSMASH 5.0.

Fig. 4. Gilliamella strains protect against M. plutonius infection through the secreted RiPPs in honeybee gut.

a Full phylogenetic tree of the Gilliamella genus based on the whole-genome sequences using the maximum-likelihood algorithm. The inner layer surrounding the genomic tree designates taxonomic annotations, while the remaining layers depict the classes of BGCs in the genome. A full tree is shown in Supplementary Fig. 5. b The cell-free supernatant of G. apis B14384-H2, W8126 inhibits the growth of M. plutonius in vitro. c Schematic of the in vivo experiments. MF bees were colonized with G. apis B14384-H2, W8126, G. apicola W8136, and B14384G12 for 7 days and then orally infected with M. plutonius (10⁶ bacterial cells per bee). d Absolute abundance of M. plutonius of different treatment groups 7 days post-inoculation with M. plutonius. The overall significance was first assessed by one-sided ANOVA (p  =  0.001). Duncan’s test was used for multiple comparisons. The letters above each bar show statistical differences among the treatments (p  <  0.05) (n = 5 bees for both groups). Box boundaries are the 25th and 75th percentiles, the horizontal line across the box is the median, and the whiskers indicate the minimum and maximum values. e Organization of the G. apis B14384H2 and W8126 strains RiPPs biosynthesis cluster. The proposed biosynthetic genes and surrounding regions are displayed. f Base peak chromatograms of extracts from G. apis B14384H2 with images of the antibacterial activities by micro-plate assay showing the bioactivity of 95–115 min fractions correlating to the portion of the chromatogram against M. plutonius. g The precursor peptide of the RiPPs was identified by MetaMiner. Post-translationally modified amino acids are shown in blue. Source data are provided as a Source Data file.