Progress 03/01/24 to 02/28/25
Outputs
Target Audience:Target audience: Beekeepers: Beekeepers were targeted because they face annual colony loss due to pathogenic infection and parasitic infestation of hives, resulting in economic loss. There is an unmet need for tools to combat these diseases in a safe manner. Beekeepers were targeted long term, through research to provide tools to improve bee health. Undergraduates: Undergraduates were targeted to provide hands on training in research and biology, one of the goals of the University, and to promote interest in pollinator research. I continued to providementorship to one undergraduate who contributed to this project, over the course of the 2024-2025school year. I have also provided this student with professional development guidance and intend to hire her as a technician in my own lab in Fall 2025. Scientists in the fields of microbiology, host-symbiont interactions, and pollination: Microbiologists were targeted because the genome engineering tools we are developing for engineering bee gut bacteria have broader applicability and have the potential to inform genome engineering tool development in other bacteria. Host-symbiont scientists were targeted because the genome engineering tools we are developing will enable new lines of investigation into host-symbiont interactions, through the easy use of reverse genetics. Pollinator (eg, honey bee) scientists were targeted because our research will enable easiser study of honey bee microbes and hopes to provide tools for improving bee health. Scientists were targeted short term and long term. Short term, through seminars, poster sessions, and publication of reserach. Long term, trhough research to provide tools to engineer and study bee gut symbionts, and to improve bee health. Changes/Problems:The original goal of the project was to engineer symbionts for therapeutic use, to directly combat bee pathogens. Last year, I developed a robust genome engineering protocol that allows knock-in and knockout of genes in the bee gut symbiont S. alvi. Following publication of this approach in mBio, I realized that this technique could be a powerful tool for doing reverse genetics in S. alvi, to explore the mechanism of how this critical pollinator interacts with the bee. As a result, I have shifted focus to leverage this genome engineering approach to study how S. alvi uses biofilms and vesicles to colonize and interact with the bee gut. S. alvi is critical in providing colonization resistance against bee pathogens. Therefore, the study of how S. alvi colonizes and interacts with the bee host is important for gaining insight into how it naturally combats bee disease. Although the immediate aims have shifted in focus into understanding the mechanism of interaction of bees and a critical symbiont, at a high level, this work still aligns with the long term goal to use engineered symbionts to improve bee health. Insight gained into how S. alvi colonizes and interacts with the bee aims to inform the future development of symbiont-based therapeutics. What opportunities for training and professional development has the project provided? Mentorship received by PJ Lariviere: While working on this project, I have received mentorship from my advisor (Moran) and co-advisor (Barrick). I meet with Dr. Moran one-on-one every two weeks, where I have a chance to receive training inexperimental design and data interpretation. I have received similar advice from Dr. Barrick in less formal settings. I present my research at both lab meetings every few months and receive higher level feedback from both advisors, as well as labmates. I have also received mentorship from both advisors during the manuscript writing process, as both advisors contribute substantially to manuscript editing and helping shape the overall story. Additional training received by PJ Lariviere: I received advancedbeekeeping training from an expert in the lab. I have learned how to install hives from scratch and how to clip wings/mark queens. Mentorship/training received by undergraduate: I am currently mentoring one undergraduate (10 hrs/week) who is performing wet lab research on this project. I have trained her in multiple research techniques, including bacterial genome engineering, bacterial fitness assays, and working with vesicles. Professional development: I have participated in multiple conferences that increased my knowledge of host-symbiont interactions and bacterial genome engineering. Conferences include the 2024Conference on Beneficial Microbes, the 2024National Postdoc Appreciation Week Research Symposium, Entomology 2024, and the 2024USDA NIFA Pollinator Health: Research and Application Program Project Directors Meeting. This year, I also applied for tenure track faculty positions.I had 17 first round Zoom interviews (UT San Antonio, University of Oklahoma, Montana State University, Loyola University Chicago SOM, Montclair State University, Rowan University, Clemson University, University of Tennessee at Knoxville, University of South Carolina, Loyola University Chicago, Mississippi State University, Case Western Reserve University, University of North Florida, University of Rhode Island, Kennesaw State University, University of Louisville, Florida Institute of Technology).I subsequently had 7 in-person interviews (University of South Carolina, University of Oklahoma, Clemson University, University of Miami, Kennesaw State University, Montclair State University, and University of Rhode Island). How have the results been disseminated to communities of interest?Research into development of tools to engineering the bacteria of bee guts and applications aimed at improving bee health have been disseminated through seminars and conferences. I spoke at the 2024 USDA NIFA Pollinator Health: Research and Application Program Project Directors Meeting, to communicate findings to NIFA stakeholders and other awardees. I spoke at the 2024 UT Austin Interdisciplinary Life Sciences Graduate Program seminar (BioTACOS), to communicate findings to multiple departments within UT. I gave 7 research seminars (University of South Carolina, University of Oklahoma, Clemson University, University of Miami, Kennesaw State University, Montclair State University, and University of Rhode Island) to communicate work I have done on the bee microbiome, during my job talks. I presented posters at Entomology 2024 and Beneficial Microbes 2024, to insect biologists and symbiosis researchers, respectively. I also gave a poster presentation at the 2024 UT Austin National Postdoc Appreciation Week Research Symposium, to communicate findings to other UT postdocs. Work describing a protocol for engineering the genome of bacterial bee gut symbionts was accepted and published at mBio. Work describing the requirement of biofilm formation for colonization by a bee gut symbiont has been deposited to a preprint server and is currently under review at AEM. What do you plan to do during the next reporting period to accomplish the goals?We have made headway towards understanding how symbionts colonize and interact with bees using biofilms and vesicles. Details of both mechanisms still remain unclear. Within the next reporting period, efforts will aim to: 1. Visualize colonization of bee guts by Snodgrassella containing or lacking StaA/B -Using fluorescence microscopy 2. Revise and resubmit manuscript (describing the role of biofilm formation in colonization) to AEM 3. Visualize where in the bee gut StaA/B localize -Using immune-electron microscopy 4. Determine the identity binding partner of StaA/B, in the bee gut -Using Co-IP and mass spec 5. Determine if knockout of vesicle formation influences bee gut colonization and nucleotide transfer -By assessing CFU counts and fluorescence of colonized symbionts -Also assess dsRNA transferred from symbiont to beeby qPCR 6. Determine if vesiculation mutants have reduced fitness in vitro -By assessing growth curve and CFU counts 7. Determine if RNA identity is impacted in vesiculation mutants -Via RNAseq 8. Submit manuscript describing how vesiculation impacts interaction with the bee
Impacts
What was accomplished under these goals?
Honey bees are critical pollinators in agriculture. Honey bees face annual colony loss due to pathogens/parasites, causing economic loss. The bee microbiome, including the species Snodgrassella alvi, contribute to the health of the bee, in part by providing colonization resistance against pathogens. Yet, the mechanisms by which S. alvi colonizes and interacts with the bee host have not been elucidated. Work this year has therefore pivoted to focus on the manner in which S. alvi uses biofilms and vesicles to colonize and interact with the bee host. In the near term, other honey bee researchers will benefit from our contributions to the field, in terms of increased understanding of how symbionts interact with honey bees. Other microbiologists and host-symbiont scientists will also benefit, as we describe interaction mechanisms that rely on universal principles within microbiology (biofilm formation and vesicle production). Longer term, commercial beekeepers and farmers stand to benefit from our research into honey bee health. Insight gained into how bee symbionts interact with their host aim to improve the next generation of probiotic-based therapeutics to combat the bee pathogens driving colony loss. Any insight gained into the development of solutions for combating bee disease has the potential to decrease colony loss, thereby improving efficiency of the pollinator industry and more broadly, food security. Goals and accomplishments: Role of biofilms in symbiont colonization of the bee gut Goal: Engineer mutant bee symbiont strains predicted to be involved in colonization -We engineered bee symbiont strains with knockouts in genes predicted to be involved adhesion/colonization Goal: Assess colonization of mutant bee symbiont adhesin strains -We tested for the ability of adhesin knockout strains to colonize bees -We found WT outcompetes adhesin knockout mutants -Many adhesin knockout mutants can mono-colonize bees -A handful of knockouts (ΔstaA, ΔstaB, ΔmltA) are unable to mono-colonize bees Goal: Assess ability of mutant adhesin strains to form biofilm -We tested for the ability of adhesin knockout strains to form biofilm in vitro -Most adhesin knockout strains had reduced (but non-zero) biofilm formation, compared to WT -Two knockouts (ΔstaA and ΔmltA) had no biofilm formation Goal: Assess ability of mutant adhesin strains to auto-aggregate -We tested for the ability of cells containing adhesin knockouts to bind to other cells -We found ΔstaA cells cannot auto-aggregate, unlike WT, which can -By SEM, ΔstaA lack cell-cell connections (normally present in WT) Goal: Assess predicted structure and conservation of bee symbiont adhesins -We predicted the structure of StaA and StaB using Alphafold -We found that StaA/B in Snodgrassella are massive proteins, substantially larger than those that have been described in other species in the literature -We also constructed a phylogeny of StaA and StaB across the Snodgrassella genus -We found that StaA and StaB are conserved across both honey bee and bumblebee-associated Snodgrassella Goal: Assess susceptibility of bee symbiont biofilm knockouts to antibiotics -We tested the susceptibility of ΔstaA (vs. WT) to two bactericidal antibiotics -We found ΔstaA is more sensitive to antibiotics (including apidaecin 1B, normally found in the bee gut) than WT Role of vesicles in bee-symbiont interactions Goal: Engineer mutant bee symbiont strains predicted to be involved in vesicle production -We engineered around a dozen strains of Snodgrassella containing knockouts in predicted vesicle production genes Goal: Quantify vesicle production in mutant bee symbiont strains -A collaborator assessed vesicle production by NTA -They found that, in a handful of strains, vesicle production is substantially reduced, compared to WT Goal: Quantify RNA content in vesicle production mutant strains -We found RNA is substantially reduced in a handful of vesicle production strains Goal: Assess biofilm formation in vesicle production mutant stains -We found biofilm formation is not reduced in vesicle production strains (compared to WT) Summary of key outcomes/accomplishments: 1. Determined that symbiont biofilm formation is required for colonization of the bee gut. 2. Described multiple adhesins (StaA, StaB) that are required for bee gut colonization. 3. Determined that the StaA adhesin is involved in auto-aggregation and protection against antibiotics. 4. Determined that StaA/B are massive, conserved proteins. 5. Wrote manuscript describing that biofilm formation is required for colonization of the bee gut (deposited to bioRxiv; under review at AEM). 6. Presented posters/oral presentations describing key findings from the above points. 7. Engineered bee gut symbiont mutants involved in vesiculation. 8. Determined that a handful of mutants have a substantial reduction in vesicle production. 9. Determined that vesicle production mutants have reduced RNA content in vesicles, but that biofilm formation is not reduced.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Lariviere PJ, Ashraf AHMZ, Navarro-Escalante L, Leonard SP, Miller LG, Moran NA, Barrick JE. 2024. One-step genome engineering in bee gut bacterial symbionts. mBio 15:e01392-24.
https://doi.org/10.1128/mbio.01392-24
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Virulence-linked adhesin drives mutualist colonization of the bee gut via biofilm formation
Patrick J. Lariviere, A. H. M. Zuberi Ashraf, Isaac Gifford, Sylvia L. Tanguma, Jeffrey E. Barrick, Nancy A. Moran
bioRxiv 2024.10.14.618124; doi: https://doi.org/10.1101/2024.10.14.618124
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
E.V.S. Motta,P.J. Lariviere,K.R. Jones,Y. Song,& N.A. Moran, Type VI secretion systems promote intraspecific competition and host interactions in a bee gut symbiont, Proc. Natl. Acad. Sci. U.S.A. 121 (44) e2414882121, https://doi.org/10.1073/pnas.2414882121 (2024).
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Progress 03/01/23 to 02/29/24
Outputs
Target Audience:Target audience: Beekeepers: Beekeepers were targeted because they face annual colony loss due to pathogenic infection and parasitic infestation of hives, resulting in economic loss. There is an unmet need for tools to combat these diseases in a safe manner. Beekeepers were targeted short term and long term. Short term, through seminar disseminating work. Long term, through research to provide tools to improve bee health. Undergraduates: Undergraduates were targeted to provide hands on training in research and biology, one of the goals of the University, and to promote interest in pollinator research. I provided mentorship to one undergraduate who contributed to this project, over the course of the 2023-2024 school year. Scientists in the fields of microbiology, host-symbiont interactions, and pollination: Microbiologists were targeted because the genome engineering tools we are developing for engineering bee gut bacteria have broader applicability and have the potential to inform genome engineering tool development in other bacteria. Host-symbiont scientists were targeted because the genome engineering tools we are developing will enable new lines of investigation into host-symbiont interactions, through the easy use of reverse genetics. Pollinator (eg, honey bee) scientists were targetedbecause our research will enable easiser study of honey bee microbes and hopes to provide tools for improving bee health. Scientists were targeted short term and long term. Short term, through seminars, poster sessions, and publication of reserach. Long term, trhough research to provide tools to engineer and study bee gut symbionts, and to improve bee health. Changes/Problems:B. apis isolation and engineering proof of concept: This project uses the bacteriaB. apis, but the strain we had in lab was mislabeled. Prior to my joining the lab, a former trainee had misnamed (and published on)a bacterial strain as B. apis.Before starting my experiments, I performed 16S rDNA sequencing, and discovered this was a different bacterial species. I therefore had to isolate B. apis from our bee hives and verify that it was the correct species. Following this, I needed to perform proof of concept work to demonstrate feasbility of engineeringB. apis. The ability to engineer "B. apis"had previously been shown and was a foundation for the research project, but once we realized this strain was misnamed, we realized we needed to demonstrate that we could engineer the real B. apis. I successfully conjugated a plasmid, demonstrating that this species is in fact capable of being engineered. From this proof of concept study, we identified key plasmid parameters (origin of replication, antibiotic resistance cassette, promoter) that facilitate successful engineering. Demonstration of feasibility of engineering B. apismeans that we will now be able to work towardsengineering it according to the aims of the project. We have also added focus to additional aspects of engineering bee gut symbionts and understanding their interaction with the honey bee host, including developing a method for easy genome engineering of bee symbionts (preprint deposited), characterizing colonization of a bee symbiont (manuscript submission targeted for June 2024), and characterizing interaction of a symbiont with the bee via extracellular vesicles. What opportunities for training and professional development has the project provided?Mentorship recieved by PJ Lariviere: While working on this project, I have received mentorship from my advisor (Moran) and co-advisor (Barrick). I meet with Dr. Moran one-on-one every two weeks, where I have a chance to recieve training in experimental design and data interpretation. I have recieved similar advice from Dr. Barrick in less formal settings. I present my research at both lab meetings every few months and recieve higher level feedback from both advisors, as well as labmates. I have also received mentorship from both advisors during the manuscript writing process, as both advisors contribute substantially to manuscript editing and helping shape the overall story. Additional training recieved by PJ Lariviere: I received formal beekeeping training from an expert in the lab. Through multiple training sessions, I have been trained on how to work with bee hives, including handling bee frames, assessing overall health of the hive, cleaning the hive, and maintaining the status of a single queen per hive. Mentorship/training received by undergraduate: I am currently mentoring one undergraduate (10 hrs/week) who is performing wet lab reserach on this project. I have trained her in multiple research techniques, including rearing honey bee larvae in lab, performing PCR, performing bacterial cell culture, and bacterial genome engineering. She has also received formal training to work with bee hives. Professional development: I have participated in multiple conferences that increased my knowledge of host-symbiont interactions and bacterial genome engineering. Conferences include ASM Microbe 2023, NSF EDGE meeting 2023, and the NSFBuild-a-Cell Workshop 2023. I also received training in curriculum development through participation in a series of Instructor Learning Community meetings, where instructors met mutliple times a semester to discuss the merits of content reduction in curriculum, using data to inform our perspectives. Finally, as the project lead on this research project, I have gained experimence managing a researchbudget. How have the results been disseminated to communities of interest?Research into development of tools to engineering the bacteria of bee guts and applications aimed at imrpoving bee health have been disseminated through seminars and conferences. I spoke at the Northern Colorado Beekeepers Association, to communicate directly to beekeepers the research that is being done to engineer the honey bee microbiome to combat disease. I gave a poster presentation at ASM Microbe 2023 to communicate a protocol we developed to engineer the genome of bacterialhoney beesymbiontsto other microbiologists. I also gave a poster presentation at NSF EDGE 2023 on work describing use of engineered honey bee symbionts to combat bee disease, with the target audience being other biologists that are developing genome engineering tools. In April 2024, I gave a deparmental seminar describing how a bacterial symbiont colonizes the honey bee, with the target audience being researchers in the molecular biosciences. Work describing use of engineered bee gut symbionts to combat Nosema infection was published in a journal article, with the target audience being other researchers and stakeholders interested in improving bee health. Work describing a protocol for engineering the genome of bacterial bee gut symbionts has been deposited to a preprint server and is currently under review at mBio. What do you plan to do during the next reporting period to accomplish the goals?We have made headway towards engineering B. apis and demonstrating colonization of larvae in our lab, but have not yet demonstrated pathogen killing. Within the next reporting period, efforts will aim to: 1. EstablishP. larvae, and eventually, Varroa,killing assays -We have had initial challenges purifying P. larvae spores, possibly due to attempting purification at too small of a scale. We will plan to scale up purification and will reach out to Dr. Jay Evans (USDA Bee Research lab), our collaborator who supplied the strains of P. larvae, for advice 2. EngineerB. apisto express effectors against Varroa and P. larvae -We do not anticipate major challenges here, as we demonstrated B. apis can be engineered 3. Test ability of WT B. apis to prevent P. larvae infection -Once we have the P. larvae infection assay up and running, we will be able to test this immediately 4. Test ability of engineered B. apis to kill P. larvae in vitro and in vivo -We may face challenges for the in vivo tests if our engineered B. apis has difficulty killing P. larvae in vitro. If this is the case, we will troubleshoot in vitro killing and test additional antimicrobial peptides for P. larvae killing.
Impacts
What was accomplished under these goals?
Honey bees are critical pollinators in agriculture. Honey bees face annual colony loss due to pathogens/parasites, causing economic loss. Bee susceptibility to disease has revealed a need for tools to combat disease, as well as investigation into how bees interact with microbes, with an aim to improve bee health. Work here has focused on development of genome engineering tools for bee symbionts, and engineering bee symbionts to combat disease and investigate how certain symbionts interact with the bee. In the near term, other honey bee researchers will profit from our contributions to the field, both in terms of increased understanding of how symbionts interact with honey bees, but also specifically through use of a simple method for engineering the genome of honey bee symbionts that we developed. Other microbiologists and host-symbiont scientists will also benefit, as the genome engineering method we developed has the potential to be used in other bacteria. Longer term, commercial beekeepers and farmers stand to benefit from our research into honey bee therapeutics, as these aim to combat the pathogens driving colony loss. Any insight gained into the development of solutions for combating bee disease has the potential to decrease colony loss, thereby improving efficiency of the pollinator industry and more broadly, food security. Goals and accomplishments: Goal: Isolate the bee symbiont B. apis -We determined we had the wrong species of symbiont isolated, so we re-isolated this symbiont from our bee hives and verified that it is the correct one. Goal: Engineering the bee symbiont B. apis -We performed a proof of concept study to demonstrate the feasibility of engineering this symbiont -We were able to successfully engineer this symbiont (by transfer of plasmid by conjugation) and showed that it could express fluorescent protein in lab -We were able to determine key parameters required for successful engineering (origin of replication, promoter, antibiotic resistance cassette) Goal: Establish larval rearing assay in lab -We successfully established the ability to rear bee larvae in our lab in 48-well plate format, a key feature necessary for performing tests on therapeutic efficacy Goal: Establish B. apis colonization protocol of larvae -We successfully established the ability to colonize larvae in our lab with the symbiont B. apis, another key feature necessary for being able to deliver therapeutics to the larvae Goals: Establish P. larvae killing assay and engineer B. apis to express effectors -We have initiated work to establish a P. larvae killing assay and to engineer B. apis to express effectors. These are in progress. Goal: Develop genome engineering protocol for bee bacterial symbionts -We have developed a simple method for engineering the genome of bacterial bee symbionts. -We describe this method in a manuscript that has been deposited to a preprint server and the manuscript is currently under review at mBio. Goal: Characterize colonization of bee by a bacterial symbiont -We have characterized the colonization of the bee by a bacterial symbiont -We find that biofilm formation is required for colonization by the symbiont S. alvi -We utilized the genome engineering protocol we developed (above) to drive this investigation -We aim to submit a manuscript describing these findings in June 2024. Goal: Characterize interaction of a bacterial symbiont with the bee -We are using reverse genetics assess how a bacterial symbiont uses vesicles to interact with the bee -We are using the genome engineering protocol we developed (above) to engineer strains to help with this goal Summary of key outcomes/accomplishments: 1. Isolated B. apis and demonstrated feasibility of engineering this symbiont 2. Established ability in our lab to rear bee larvae and colonize with B. apis 3. Initiated work to establish P. larvae killing assay and engineer B. apis to express effectors 4. Wrote manuscript describing method for engineering the genomes of diverse bee symbionts (under review) 5. Finished collecting data for 2nd manuscript, describing colonization of bee by a bacterialsymbiont (submission target: June 2024)submitted 6. Presented posters/oral presentations describing key findings from both manuscripts above 7. Initiated work to characterize the interaction of a bacterial symbiont through vesicles
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Lariviere, P. J. et al. Single-step genome engineering in the bee gut symbiont Snodgrassella alvi. BioRxiv Prepr. Serv. Biol. 2023.09.19.558440 (2023) doi:10.1101/2023.09.19.558440. Under review at mBio.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Huang, Q., Lariviere, P. J., Powell, J. E. & Moran, N. A. Engineered gut symbiont inhibits microsporidian parasite and improves honey bee survival. Proc. Natl. Acad. Sci. 120, e2220922120 (2023).
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