Performing Department
(N/A)
Non Technical Summary
Infections of deformed wing virus, Israeli acute paralysis virus, and other viruses threaten honey bee colony health and pollination services that are critical for agriculture. One member of our team has demonstrated that feeding honey bees certain peptides can protect them from transmitting of viruses by blocking the gut receptors that viruses use for entering bee cells. However, it is challenging to continuously deliver a sufficient dose to achieve protection by feeding peptides to bees. Honey bees naturally harbor a beneficial community of bacterial symbionts in their guts. Another member of our team has created a toolkit for genetically engineering these symbiotic bacteria so that they can continuously produce biomolecules within bees. The final member of our team has shown how natural peptide secretion systems can be repurposed to engineer diverse bacteria to secrete a wide variety of peptides. In this project, we will combine our expertise to test how effective continuous secretion of gut-binding peptides by gut symbionts is at protecting honey bees against viruses. In addition, we will characterize how molecules disperse within the bee digestive tract and how engineered bee gut symbionts spread among bees, both within and between hives. This information will generally inform future efforts to use gut symbionts to continuously dose bees with different types of bioactive molecules to combat current and emerging threats to pollinator health.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
Infections of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and other viruses threaten honey bee colony health and contribute to high rates of overwintering losses. In prior work, members of our team demonstrated that feeding honey bees peptides that mimic viral capsid structures can protect honey bees from infection by blocking the gut receptors that these viruses use for entering bee cells. However, it is challenging to continuously deliver a sufficient dose for protection by feeding peptides to bees. Alternative methods for delivering peptides could increase the efficacy of protection and make it more economical to use this technology to manage viruses.Honey bees have a co-evolved gut microbiome that consists of a conserved set of bacteria that are important for their health. Other members of our team have engineered symbiotic bacteria in the bee gut microbiome (BGM) to continuously produce double-stranded RNA molecules to induce RNA interference (RNAi) responses that protect bees from parasites and pathogens. Separately, we have shown how microcin type 1 secretion systems (T1SS) can be repurposed to engineer bacteria to secrete a wide variety of peptides. With the proposed studies, we will extend the molecular modalities available for paratransgenic protection of honey bees to include peptide secretion, and we will determine how effective this strategy is for protecting honey bees from viral infections.The objectives of this proposal are to:1. Secrete peptides from bee gut bacteria and characterize peptide dispersal1.1 Assess the native T1SS peptide secretion capacity of bee gut symbionts1.1.1 Screen native Gilliamella microcin T1SSs using the zone of inhibition assay1.1.2 Demonstrate and quantify protective peptide secretion1.2 Optimize heterologous T1SS peptide secretion from BGM bacteria1.2.1 Create modular parts for more flexible assembly of peptide secretion systems1.2.2 Promoter parts for controlling and optimizing peptide expression1.2.3 Integrate secretion systems into the chromosomes of S. alvi and other BGM bacteria2. Protect bees from viruses through paratransgenic peptide secretion2.1 Test paratransgenic protection of newly emerged worker bees2.1.1 Screen additional gut-binding peptides from phage-display libraries for virus protection2.1.2 Test how well paratransgenic peptide secretion protects honey bees from viruses2.2 Examine the effectiveness of paratransgenic protection in hives2.2.1 Test transmission and maintenance of engineered BGM bacterium within a hive2.2.2 Test intercolony transmission of engineered BGM bacteriaIn addition to testing a specific innovative strategy for protecting bees against viruses, this proposal will answer basic science questions about how molecules, including those naturally released by gut symbionts, mix within the bee's alimentary canal and how engineered bacteria and DNA sequences would spread within bee hives and the environment if they were used in the field. This information will generally inform future efforts to use gut symbionts to continuously dose bees with different types of bioactive molecules to combat emerging threats to pollinator health.
Project Methods
1.1 Assess the native T1SS peptide secretion capacity of bee gut symbiontsWe will use a zone of inhbition assay to screen ≥10 Gilliamella strains for killing of E. coli bacterial prey via their native type I secretion system (T1SS) of MccV with the E. coli secretion system signal peptide. For promising strains, we will engineer an RSF1010 plasmid system to encode for IPTG inducible expression of MccV fused to the native signal sequences of endogenous Gilliamella microcins. If we do not have promising results from 3+ strains, including at least one G. apis and one G. apicola, we will clone Gilliamella Type I secretion systems into the plasmid so that addition of IPTG induces expression of both the native export system and its MccV cargo. In all cases, each strain will be spotted on a lawn of prey E. coli. Active export will be scored by measuring the size of the zone of inhibition. To quantitatively measure peptide export through Gilliamella type I systems in liquid medium, we will add a V5 epitope tag to the protective peptides and use blotting.1.2 Optimize heterologous T1SS peptide secretion from BGM bacteriaPreviously, we showed that RSF1010 plasmids replicate in various bee gut bacteria. To increase the flexibility with which we can change secretion signals, peptides, promoters, and secretion systems, we will create standardized genetic parts for components of T1SSs that are compatible with our BTK Golden Gate assembly scheme. The PCAT and MFP will be combined as one Type 3 part. Signal sequences and secreted peptides will be integrated by splitting Type 3 parts in Type 3a and Type 3b parts. We will also experiment with making a two-plasmid system in which the T1SS and its cargo are encoded on different plasmids, using the pBBR1 origin that has been tested in S. alvi and Bartonella apis.We will test optimized inducible expression systems from the Marionette collection by creating Type 2 promoter parts and Type 4 parts that constitutively express the corresponding regulatory proteins. We will characterize these systems in Snograssella and Gilliamella strains in vitro and then in bees using these parts to regulate expression of a Type 3 GFP part. Then, Type 3 parts encoding protective peptides of interest or the secretion system can be assembled in place of GFP. We will also use a Type I-F CRISPR-associated transposase system to site-specifically integrate these genetic payload into the chromosomes of bee gut symbionts to make the system more genetically stable and eliminates the need to add antibiotics.1.3 Examine dispersal of peptides secreted by different BGM bacteriaWe will use immunostaining of epitope-tagged gut-binding peptides to detect them. Bee gut symbionts engineered to secrete peptides will be allowed to fully colonize newly emerged workers for 5 days post-inoculation. Then we will sacrifice bees, dissect their intact guts, fix them, immunostain, and image. Peptide localization may be affected by mixing that occurs as food and water traverse the alimentary tract, so we will also examine bees that have been starved for 8 hours to try to minimize this disturbance. Finally, we will contrast dispersal of symbiont-secreted peptides with oral peptide delivery by hand-feeding bees at the same developmental stage synthetic versions of the same peptides. As a complement to immunostaining, we will use in situ scanning mass spectrometry to directly detect peptides and examine their movement and stability in bees.2.1 Test paratransgenic protection of newly emerged worker beesTo establish appropriate experimental conditions, 10 replicate acrylic cages, each containing 24 day-old bees will be housed in an incubator. We will age these bees to five-days. Then, the caged workers will be anesthetized and individually fed a sucrose solution containing IAPV or DWV with or without a high concentration of the gut-binding peptide or a control, non-binding peptide. An additional control of mock infected bees fed sucrose solution with no virus will be included. Honey bee mortality will be monitored daily by removing and counting all dead bees from each cage for the duration of the experiment (7 days). Differences in survival will be assessed using logrank tests. After a period of 36 h, a subset of bees (n=6) will be collected from each cage and tested by RT-qPCR for virus titer using established protocols. The dose of virus inoculum and concentration of synthetic peptide employed will be optimized in subsequent experiments. We anticipate testing a total of five gut binding peptides. Statistical analyses to assess for significant differences in virus titer between treatments will be conducted using ANOVA.We will use the same experimental design to test peptides secreted by engineered gut bacteria, except we will colonize 30 microbiota-deficient newly emerged worker bees per replicate and feed them on sucrose solution containing antibiotic to select for the engineered strain and plasmid maintenance. We will colonize these newly emerged worker bees with gut microbes that express both GFP and secrete peptides. Our controls will be: (i) bees that are not inoculated with bacteria and (ii) bees inoculated with the same symbiont except with a mutated signal sequence so peptide is not secreted. Bacterial colonization will be verified by sacrificing bees (n=6) per group prior to virus inoculation, pulling their guts, plating on selective media, and counting colony forming units. We will also verify that uncolonized bees are microbiota-deficient using 16S rRNA gene qPCR with universal primers. We will monitor survival and collect bees (n=6) 36 h after virus inoculation to test if virus titers are affected.2.2 Examine the effectiveness of paratransgenic protection in hivesWe will utilize triple cohort colonies, consisting of 700 day old, 700 week old, and 700 two week old bees, housed in 5-frame nucleus hive boxes. Two non-focal hives will be located one meter away on either side of the focal hive within the same flight chamber. Bees will be marked to indicate age, treatment, and home colony. We will conduct five complete replicates of these experiments, each using a different set of three hives and a different engineered symbiont. We will test different bee gut species as hosts for peptide secretion, including at least one experimental replicate with an engineered G. apicola, one with G. apis, and one with S. alvi.For each replicate, we will inoculate 200 newly emerged workers with an engineered bacterial symbiont expressing GFP and secreting a gut-binding peptide. These bees will be reared to five days of age in cages in incubators in the lab while fed on sucrose solution with antibiotic so the engineered bacteria fully colonize each bee. We will validate colonization on a sample of bees (n=6). We will then mark the colonized bees with paint and introduce them into the focal hive in "push-in" cages for one day to increase acceptance. After they have mixed with the focal colony for 48 hours, we will orally infect this hive with a moderate dose of virus.Colonies will be monitored for 21 days. At 7 and 14 days, a subset of foragers and in-hive workers from the focal colony, including at least 50 bees that were inoculated with engineered bacteria colony and 80 that were not (balanced over age groups), will be collected. At the end of the experiment, we will collect the entire remaining population of each colony. Virus levels will be assessed by RT-qPCR at all three timepoints. We will track maintenance and dissemination of engineered bacteria and DNA in the sampled bees by using 16S rRNA gene metagenomics and specific qPCRs with different primer sets to monitor overall bacterial abundance, engineered strain abundance, and the copy numbers of engineered sequences. We will also monitor for transmission of the engineered BGM bacteria to the adjacent hives that were not treated with virus by sampling 240 of their workers at the same three time points.