Source: THE UNIVERSITY OF TEXAS AT AUSTIN submitted to
ENGINEERING BACTERIA TO COMBAT BEE PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1030005
Grant No.
2023-67012-39356
Project No.
TEXW-2022-09624
Proposal No.
2022-09624
Multistate No.
(N/A)
Program Code
A1113
Project Start Date
Mar 1, 2023
Project End Date
Feb 28, 2025
Grant Year
2023
Project Director
Lariviere, P. J.
Recipient Organization
THE UNIVERSITY OF TEXAS AT AUSTIN
101 EAST 27TH STREET STE 4308
AUSTIN,TX 78712-1500
Performing Department
(N/A)
Non Technical Summary
The honey bee is one of the most ecologically and economically important pollinators worldwide. Perhaps the biggest threat facing bees (both in industry and nature) is pathogen-borne disease. Two of the most prominent bee diseases, American foulbrood and Varroa mite infection, have been difficult to contain, contributing to over $300 million in hive replacement costs per year and declining food security. Current therapeutics lack specificity, efficacy, and tolerable side-effect profiles. Fortunately, synthetic biology offers a solution through the use of symbiont engineering. Bee symbionts have previously been genetically modified to combat Varroa infections in individual adult bees. However, larval and hive-level infections remain untamed. Further, use of symbionts to combat foulbrood has not yet been attempted. In this proposal, I aim to engineer hive-associated bacteria to kill bee pathogens. The natural hive-associated bacterium Bombella apis will be engineered to express antimicrobial bacteriocins to target the foulbrood-causing agent Paenibacillus larvae. Additionally, B. apis will be used to kill larvae-associated Varroa mites through symbiont-mediated RNAi. Efficacy for both therapeutics will be measured in terms of effective pathogen killing and improvement of bee larval survival and health.This research project aims to provide novel therapeutics in the fight against two of the biggest bee pathogens, leading to improved efficiency of natural and commercial crop pollination. This work directly addresses NIFA AFRI Farm Bill Priority Area PHPPP, Program Area 1f. Pollinator Health: Research and Application (A1113) through the development of new therapeutics to promote pollinator health. Finally, note that research using engineered organisms will becarried out in a laboratory (not outdoors), according tostandard biosafety procedure.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31130101100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3010 - Honey bees;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Goal: Engineer bee larval symbiotic bacteria tocombat larval bee disease.Objective 1: Engineer the bee larval symbiont Bombella apisto be able to inhibit the growth of the causitive agent of American Foulbrood,Paenibacillus larvae, in a larval disease model. This objective will be achieved by engineering B. apisto secreteP.larvae-specific antimicrobial bacteriocins that have been identified through a bioinformatic pipeline.Objective 2:Engineer Bombella apisto kill bee larvae-associatedVarroa mites in a larval disease model. This objective will be achieved by engineering B. apisto secreteVarroa-specific dsRNA, using dsRNA constructs previously shown to killVarroamites in adult bees.
Project Methods
The objectives (1. EngineerB. apisto killP. larvae; 2. EngineerB. apisto kill bee larval-associatedVarroamites) will each be divided into 4 four phases:Phase 1: Plasmid/strain engineeringDesign and construct plasmids to expressP. larvae-targeting bacteriocins, export machinery, and immunity protein (Obj. 1)Conjugate bacteriocin- and dsRNA-expressing plasmids intoB. apiswith a conjugation competentE. colistrain (Obj. 1&2)Phase 2:in vitrotestingTest bacteriocin/dsRNA expression by B. apis(Obj. 1&2)Test bacteriocin/dsRNA secretion byB. apis(Obj. 1&2)Test bacteriocin activity againstP. larvaein vitro (Obj. 1)Test dsRNA activity against Varroamitesin vitro(Obj. 2)Phase 3: Test effectors efficacyin infection models (in 96 well plates)Quantify pathogen killing when exposed to treatment16s rRNA qPCR (Obj. 1)Mite mortality quantification (Obj. 2)Monitor pathogen activity (Obj. 1)Holst milk testMonitor health of treated bee larvae infected withP. larvae (Obj. 1)Quantify bee larval survivalMonitor marker of bee larval health (def-1 levels)Check for bee larval dysbiosisMonitor health of treated bee larvae infected with Varroamites (Obj. 2)Quantify bee larval survivalMonitor marker of bee larval health (relishlevels)Quantify the presence of DWV in adult bees (by qPCR)Score adult bees for deformed wingsPhase 4: Performin situeffector testing (in hive frames)Quantify pathogen killing when exposed to treatment16s rRNA qPCR (Obj. 1)Mite mortality quantification (Obj. 2)Monitor health of treated bee larvae infected withP. larvae(Obj. 1)Quantify bee larval survivalMonitor marker of bee larval health (def-1 levels)Check for bee larval dysbiosisMonitor health of treated bee larvae infected withVarroamites (Obj. 2)Quantify bee larval survivalMonitor marker of bee larval health (relishlevels)Quantify the presence of DWV in adult bees (by qPCR)Score adult bees for deformed wingsDistinctionsfrom previous work:Utilization of larval symbiont to deliver effectorsExpression of pathogen-specific bacteriocins to kill a bacterial pathogen (P. larvae)Targeting of larval-specific Varroamites using symbiont-delivered (B. apis) dsRNAVarroamites have previously been targeted with symbiont delivered dsRNA in adultbees, using an adult-specific symbiont (S. alvi)The use of a larval-specific symbiont, to target larval-specific Varroamite infections is novelMeans of communication:Publication of research in scientific journalsPresentation of research via poster/oral presentations at conferencesTimeline:Four months will be allocated for the completion of each Objective's phase (8 phases, across 2 Objectives = 2 years).Evaluation of this research's impact on the target audience will be conducted quarterly according to the following criteria:Alignment with the timeline scheduleAchievement of key research goals according to the timelineKey challenges faced and overcomeQuantity and impact of relevant manuscriptsFeedback will be solicited (and used to update the research plan) at the following venues:Quarterly meetings with mentors and collaboratorsYearly meeting with advisory committeeConsisting of internal (mentors/collaborators) and external stakeholders