Toll receptor ligand Spätzle 4 responses to the highly pathogenic Enterococcus faecalis from Varroa mites in honeybees.

Honeybees play a major role in crop pollination, which supports the agricultural economy and international food supply. The colony health of honeybees is threatened by the parasitic mite Varroa destructor, which inflicts physical injury on the hosts and serves as the vector for variable viruses. Recently, it shows that V. destructor may also transmit bacteria through the feeding wound, yet it remains unclear whether the invading bacteria can exhibit pathogenicity to the honeybees. Here, we incidentally isolate Enterococcus faecalis, one of the most abundant bacteria in Varroa mites, from dead bees during our routine generation of microbiota-free bees in the lab. In vivo tests show that E. faecalis is only pathogenic in Apis mellifera but not in Apis cerana. The expression of antimicrobial peptide genes is elevated following infection in A. cerana. The gene-based molecular evolution analysis identifies positive selection of genes encoding Späetzle 4 (Spz4) in A. cerana, a signaling protein in the Toll pathway. The amino acid sites under positive selection are related to structural changes in Spz4 protein, suggesting improvement of immunity in A. cerana. The knock-down of Spz4 in A. cerana significantly reduces the survival rates under E. faecalis challenge and the expression of antimicrobial peptide genes. Our results indicate that bacteria associated with Varroa mites are pathogenic to adult bees, and the positively selected gene Spz4 in A. cerana is crucial in response to this mite-related pathogen.

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

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.

Engineered symbiotic bacteria interfering Nosema redox system inhibit microsporidia parasitism in honeybees.

Nosema ceranae is an intracellular parasite invading the midgut of honeybees, which causes serious nosemosis implicated in honeybee colony losses worldwide. The core gut microbiota is involved in protecting against parasitism, and the genetically engineering of the native gut symbionts provides a novel and efficient way to fight pathogens. Here, using laboratory-generated bees mono-associated with gut members, we find that Snodgrassella alvi inhibit microsporidia proliferation, potentially via the stimulation of host oxidant-mediated immune response. Accordingly, N. ceranae employs the thioredoxin and glutathione systems to defend against oxidative stress and maintain a balanced redox equilibrium, which is essential for the infection process. We knock down the gene expression using nanoparticle-mediated RNA interference, which targets the γ-glutamyl-cysteine synthetase and thioredoxin reductase genes of microsporidia. It significantly reduces the spore load, confirming the importance of the antioxidant mechanism for the intracellular invasion of the N. ceranae parasite. Finally, we genetically modify the symbiotic S. alvi to deliver dsRNA corresponding to the genes involved in the redox system of the microsporidia. The engineered S. alvi induces RNA interference and represses parasite gene expression, thereby inhibits the parasitism significantly. Specifically, N. ceranae is most suppressed by the recombinant strain corresponding to the glutathione synthetase or by a mixture of bacteria expressing variable dsRNA. Our findings extend our previous understanding of the protection of gut symbionts against N. ceranae and provide a symbiont-mediated RNAi system for inhibiting microsporidia infection in honeybees.

Stably transmitted defined microbial community in honeybees preserves Hafnia alvei inhibition by regulating the immune system.

The gut microbiota of honeybees is highly diverse at the strain level and essential to the proper function and development of the host. Interactions between the host and its gut microbiota, such as specific microbes regulating the innate immune system, protect the host against pathogen infections. However, little is known about the capacity of these strains deposited in one colony to inhibit pathogens. In this study, we assembled a defined microbial community based on phylogeny analysis, the 'Core-20' community, consisting of 20 strains isolated from the honeybee intestine. The Core-20 community could trigger the upregulation of immune gene expressions and reduce Hafnia alvei prevalence, indicating immune priming underlies the microbial protective effect. Functions related to carbohydrate utilization and the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS systems) are represented in genomic analysis of the defined community, which might be involved in manipulating immune responses. Additionally, we found that the defined Core-20 community is able to colonize the honeybee gut stably through passages. In conclusion, our findings highlight that the synthetic gut microbiota could offer protection by regulating the host immune system, suggesting that the strain collection can yield insights into host-microbiota interactions and provide solutions to protect honeybees from pathogen infections.

Bartonella choladocola sp. nov. and Bartonella apihabitans sp. nov., two novel species isolated from honey bee gut.

Bartonella is one of the noncore bacterial genera in the honey bee (Apis mellifera) gut. So far, only one species, Bartonella apis, has been described from the honey bee gut. Previous analyses based on the genomic information of isolates and metagenome-assembled genomes suggested the existence of multiple Bartonella species in the bee guts. Here, 10 strains were isolated and characterized from the gut of A. mellifera from Jilin Province, China. New isolates shared ＞95% 16S rRNA gene sequence similarity with other species of the genus Bartonella. Phylogenetic analysis revealed that new isolates clustered with other type strains of Bartonella, and the bee gut Bartonella could be classified into three clades. The in silico DDH and average nucleotide identity values between strains of different clusters from the honey bee gut are 29.1-32.5% and 87.6-89.3%, all below the recommended 70.0% and 95% cutoff points. Cells are Gram-staining-negative rods and can grow on the surface of Brain Heart Infusion agar plates supplemented with defibrinated sheep blood in an aerobic environment with 5% CO2 at 35-37 °C. Strains from different species varied in both phenotypic and chemotaxonomic characterizations. Comparative genomic analysis indicated that B. choladocola had unique sets of genes encoding invasin, representing the potential for this species to both live as a gut symbiont and also as an erythrocytic pathogen. Thus, we propose two novel species Bartonella choladocola sp. nov. whose type strain is W8125T(=JCM 35030T = ACCC 62057T), and Bartonella apihabitans sp. nov. whose type strain is W8097T(=JCM 35029T = ACCC 62056T).

Geographical resistome profiling in the honeybee microbiome reveals resistance gene transfer conferred by mobilizable plasmids.

BACKGROUND: The spread of antibiotic resistance genes (ARGs) has been of global concern as one of the greatest environmental threats. The gut microbiome of animals has been found to be a large reservoir of ARGs, which is also an indicator of the environmental antibiotic spectrum. The conserved microbiota makes the honeybee a tractable and confined ecosystem for studying the maintenance and transfer of ARGs across gut bacteria. Although it has been found that honeybee gut bacteria harbor diverse sets of ARGs, the influences of environmental variables and the mechanism driving their distribution remain unclear. RESULTS: We characterized the gut resistome of two closely related honeybee species, Apis cerana and Apis mellifera, domesticated in 14 geographic locations across China. The composition of the ARGs was more associated with host species rather than with geographical distribution, and A. mellifera had a higher content of ARGs in the gut. There was a moderate geographic pattern of resistome distribution, and several core ARG groups were found to be prevalent among A. cerana samples. These shared genes were mainly carried by the honeybee-specific gut members Gilliamella and Snodgrassella. Transferrable ARGs were frequently detected in honeybee guts, and the load was much higher in A. mellifera samples. Genomic loci of the bee gut symbionts containing a streptomycin resistance gene cluster were nearly identical to those of the broad-host-range IncQ plasmid, a proficient DNA delivery system in the environment. By in vitro conjugation experiments, we confirmed that the mobilizable plasmids could be transferred between honeybee gut symbionts by conjugation. Moreover, "satellite plasmids" with fragmented genes were identified in the integrated regions of different symbionts from multiple areas. CONCLUSIONS: Our study illustrates that the gut microbiota of different honeybee hosts varied in their antibiotic resistance structure, highlighting the role of the bee microbiome as a potential bioindicator and disseminator of antibiotic resistance. The difference in domestication history is highly influential in the structuring of the bee gut resistome. Notably, the evolution of plasmid-mediated antibiotic resistance is likely to promote the probability of its persistence and dissemination. Video Abstract.

Precise Thermal Regulation of Engineered Bacteria Secretion for Breast Cancer Treatment In Vivo.

For the biomedical application of engineered bacteria, strictly regulating the function of engineered bacteria has always been the goal pursued. However, the existing regulation methods do not meet the needs of the in vivo application of engineered bacteria. Therefore, the exploration of the precise regulation of engineered bacteria is necessary. Herein, heat-sensitive engineered bacteria that can respond to thermal stimuli within 30 min were constructed, and the precise control of functions was verified in the intestines of various model organisms (including C. elegans, bees, and mice). Subsequently, heat-sensitive engineered bacteria were shown to colonize the mouse tumor microenvironment. Finally, thermal stimulation was proven to control engineered bacteria to produce the therapeutic protein tumor necrosis factor α (TNF-α) in the tumor. After three heat stimulation treatments, the growth of the tumor was significantly inhibited, suggesting that heat can be used as a strategy to precisely control engineered bacteria in vivo.

Specific Strains of Honeybee Gut Lactobacillus Stimulate Host Immune System to Protect against Pathogenic Hafnia alvei.

Honeybee gut microbiota plays an important role in host physiology and metabolism. Recent studies have shown that the influence of the resident microorganisms in the regulation of honeybee immune system is profound, which protects against the pathogen Serratia marcescens. However, only few of the core gut members in the regulation of immune functions have been studied. Here, we explored how different bee gut bacterial species aided in the clearance of the pathogenic Hafnia alvei, which causes bee septicemia with a high mortality rate. We found that both Gilliamella apicola W8136 and Lactobacillus apis W8172 protect honeybees from the opportunistic pathogen, while two other strains from Gilliamella and Lactobacillus did not affect the invasion of H. alvei. Transcriptomic analysis revealed that gut species induced different expression profiles in the gut. Specifically, two regulator genes from the Toll pathway, PGRP-S3 recognizing Gram-positive and Spätzle that bind to the Toll protein for the downstream signal transduction, were elevated by L. apis. Correspondingly, multiple genes encoding antibacterial proteins were also stimulated by L. apis. Interestingly, we found an increased expression of apidaecin, which also exhibited a high in vitro inhibitory effect on H. alvei. To elucidate the difference of strains in the host's immune regulation, comparative genomic analyses indicate that the S-layer proteins unique to L. apis are potentially involved in honeybee Toll signaling and the activation of antibacterial protein production. IMPORTANCE Honeybees are essential pollinators supporting global agricultural economies and food supplies. Recent honeybee decline has been linked to several factors, while pathogen infection is considered one of the most significant contributing factors. Although a limited number of bacterial pathogens have been identified, Hafnia alvei is one of the pathogens causing septicemia in adult bees. In this study, we showed that two bee gut members, Gilliamella and Lactobacillus, can clear H. alvei from invasion. Mono-colonization of specific strains can stimulate the host Toll signaling pathway and the downstream expression of AMPs. Specifically, apidaecin upregulated by the gut symbionts is more effective against the pathogen. Moreover, our genomic analysis suggests that the surface-layer proteins specific to Lactobacillus strains are an important driver of Toll signaling, highlighting the variation of bee gut strains in regulating the host immune system.

Honey bee genetics shape the strain-level structure of gut microbiota in social transmission.

BACKGROUND: Honey bee gut microbiota transmitted via social interactions are beneficial to the host health. Although the microbial community is relatively stable, individual variations and high strain-level diversity have been detected across honey bees. Although the bee gut microbiota structure is influenced by environmental factors, the heritability of the gut members and the contribution of the host genetics remains elusive. Considering bees within a colony are not readily genetically identical due to the polyandry of the queen, we hypothesize that the microbiota structure can be shaped by host genetics. RESULTS: We used shotgun metagenomics to simultaneously profile the microbiota and host genotypes of bees from hives of four different subspecies. Gut composition is more distant between genetically different bees at both phylotype- and "sequence-discrete population" levels. We then performed a successive passaging experiment within colonies of hybrid bees generated by artificial insemination, which revealed that the microbial composition dramatically shifts across batches of bees during the social transmission. Specifically, different strains from the phylotype of Snodgrassella alvi are preferentially selected by genetically varied hosts, and strains from different hosts show a remarkably biased distribution of single-nucleotide polymorphism in the Type IV pili loci. Genome-wide association analysis identified that the relative abundance of a cluster of Bifidobacterium strains is associated with the host glutamate receptor gene specifically expressed in the bee brain. Finally, mono-colonization of Bifidobacterium with a specific polysaccharide utilization locus impacts the alternative splicing of the gluR-B gene, which is associated with an increased GABA level in the brain. CONCLUSIONS: Our results indicated that host genetics influence the bee gut composition and suggest a gut-brain connection implicated in the gut bacterial strain preference. Honey bees have been used extensively as a model organism for social behaviors, genetics, and the gut microbiome. Further identification of host genetic function as a shaping force of microbial structure will advance our understanding of the host-microbe interactions. Video abstract.