Progress 07/01/23 to 06/30/24
Outputs
Target Audience:Dr. Choi presented an oral presentation and a poster at the CRWAD (Conference of Research Workers in Animal Disease) conference respectively 2024 and 2025. Part of this work was also presented at the International Society for Prebiotics and Probiotics (ISAPP) in Cork, Ireland. The work was also presented at the MicroERA symposium at the University of Illinois at Urbana-Champaign. In the meetings, the audience was represented by representatives from industry working in animal health, academics working in animal health or related areas, and undergraduate, graduate and postdoctoral trainees. Within the Department, weekly updates of the work was presented at our lab meeting, where all lab members, undergraduate and graduate students are present. Changes/Problems:The activity of the lysins remains underwhelming, which in part can be attributed to low levels of recombinant protein release by the engineered probiotic. We already have laid a strong scientific foundation for an alternative strategy, which is the use of our partly characterized lytic-switch, which leads to 10-100-fold increased lysis. Evidently, we predict that this increased lysis will lead to increased release of antimicrobials that have been accumulated inside the engineered probiotic. A major advantage of our approach is that lysis can be delayed. In other words, we can flip the lysis switch, and the cells lyse ~3 hours later. This opens up exciting opportunities for therapeutic delivery. In the upcoming period, we will investigate this application, which we expect will accomplish our objective to have developed an engineered probiotic that releases (enough) antimicrobial to lead to S. suis killing. What opportunities for training and professional development has the project provided?Dr. Choi, the postdoctoral trainee funded by this award: - gave oral presentation at the 2024 CRWAD meeting; - was invited for a job interview for a faculty position - continues to meet 1:1 with her advisor, Dr. van Pijkeren, to continue to build on efforts to realize Dr. Choi's long-term plan (obtain a faculty position). How have the results been disseminated to communities of interest?- oral and poster presentation CRWAD; - oral presentation International Society for Prebiotics and Probiotics in Cork, Ireland - oral presentation to early career scientists at the MicroERA event (MicroERA = Microbial Early Career Research Association) - peer-reviewed publication in Appl. Env. Microbiol. What do you plan to do during the next reporting period to accomplish the goals?After we observed that the antimicrobial activity of multiple phage lysins was below par, we hypothesized that not enough recombinant protein was released. To address this, we invested in the development of a strain that increases the release of recombinant protein. To this end, we have laid a strong foundation as we have identified a 'lytic-switch' that boosts the release of recombinant protein. In the upcoming year, which would be under a No Cost Extension which we recently submitted, we will continue these optimization studies, which we predict will increase the release of therapeutic protein and thereby the number of microbes to be killed. This will put us in the position to meet our objective of the development of a strain that releases an antimicrobila that kills S. suis, and we expect these developments will result in a peer-reviewed publication.
Impacts
What was accomplished under these goals?
We finalized the development of a bioluminescent reporting system, which was published. The delivery vehicle of antimicrobials encodes multiple adhesins, which presents the opportunity to result in long-term colonization. To prevent this, we developed a next-gen probiotic platform that has reduced colonization potential. A strain was developed in which nine genes were inactivated, and the strain has reduced colonization potential yet maintained the therapeutic potential. This work was published in Appl. Env. Microbiol. and we filed for a patent. This work was performed in parallel with our efforts to optimize the production of lysins and to assess their antimicrobial effect. The killing-effects observed were low to modest, and our efforts to enhance the killing effectivity to combine this with reuterin (a natural antimicrobial produced by L. reuteri) did not further boost the antimicrobial activity. Thus, further optimization is required and these studies are initiated and we have a solid foundation established (see plan for future reporting period).
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Alexander LM, Khalid S, Gallego-Lopez GM, Astmann TJ, Oh J-H, Heggen M, Huss P, Fisher R, Mukherjee A, Raman S, Choi IY, Smith MN, Rogers CJ, Epperly MW, Knoll LJ, Greenberger JS, van Pijkeren J-P. Development of a Limosilactobacillus reuteri therapeutic delivery platform with reduced colonization potential. Appl Environ Microbiol. 2024 Nov 20;90(11):e0031224. doi: 10.1128/aem.00312-24. Epub 2024 Oct 31. PMID: 39480094; PMCID: PMC11577788.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Choi et al. Bioluminescent monitoring of recombinant lactic acid bacteria and their products (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
van Pijkeren. Development of L. reuteri as a therapeutic delivery vehicle. (oral presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
van Pijkeren. Therapeutic delivery by engineered probiotics.
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:Dr. Choi's Abstract was selected to be presented at the 2023 Gordon Research Conference on Lactic Acid Bacteria, hosted in July 2023 in Ventura, CA. Here, the work was presented to a broad audience interested in lactic acid bacteria genetics, physiology, and engineering. The progress was also presented at weekly lab meetings where all lab members, and rotating undergraduate and graduate students attend. Dr. Choi also presented in our Departmental seminar. In addition, in this reporting period, Dr. Choi published a first author paper in mBio, which was selected by the Editor to be featured on the main page / cover. Changes/Problems:The activity of the lysins is somewhat underwhelming, and we therefore require optimizing and performing additional screens. However, no major issues have been identified so-far. What opportunities for training and professional development has the project provided?Dr. Choi, the postdoctoral trainee funded by this award: - attended the Gordon Research Conference in June 2023 - presented a poster at the GRC - spoke at a conference in Korea (by invitation) - has 1:1 meetings with her PI, Dr. van Pijkeren, who provides career advice and developed a plan with achievable goals to accomplish Dr. Choi's long-term and short-term goals How have the results been disseminated to communities of interest?- poster presentation GRC Lactic Acid Bacteria; - oral presentation Symposium in Korea: - publication in mBio, which was selected by the Editor to be featured on the website/cover of mBio What do you plan to do during the next reporting period to accomplish the goals?- continue the development of chimeric lysins; - screen a large library of S. suis isolates; - multiplex lysin production
Impacts
What was accomplished under these goals?
1. We expanded the strain collection of engineered probiotics, and we havegenerated multiple strains ofL. reuteri that are engineeredto release a recombinant phage lysin; 2. We initiated killing assays wiht recombinant lysins but the killing activity is low to modest. We are currently investigating if co-treatment with the natural antimicrobial reuterin increases the killing activity.3. To optimize protein production, high-throughput quantification is required. We now have finalized the development and optimization ofa broadly-applicableluminescent tagging system that allows in vitro, in vivo and in situ detection of recombinant bacteria and recombinant proteins, which was recently accepted for publication in mBio.4. We requested a large S. suis strain database from the University of Montreal to test our recombinant lysins and the MTA is being finalized.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Choi IY, Oh JH, Wang Z, van Pijkeren JP. Bioluminescent monitoring of recombinant lactic acid bacteria and their products. mBio. 2023 Sep 5:e0119723. doi: 10.1128/mbio.01197-23. Epub ahead of print. PMID: 37668408.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Poster presentation at Gordon Research Conference on Lactic Acid Bacteria, Ventura, CA
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
In vitro, in situ, and in vivo monitoring of recombinant lactic acid bacteria and their products. KoSFoP 44th International Symposium and Annual Meeting, REPUBLIC OF KOREA
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:We disseminated knowledge to targeted audiences through presentations at professional and technical meetings, on- and off-campus, including seminars and classroom instruction contributions. The target audience was typically scientists from the academic, private, and public sector, including students, and sometimes support staff affiliated with attending groups. Ultimately, publications in appropriate peer-reviewed journals and theses/dissertations will sustain dissemination of knowledge resulting from this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Choi, the postdoctoral researcher leading this project, has received one-on-one training from the PI, Dr. van Pijkeren. Dr. Choi was coached to improvd her presentation skills and writing skills. Dr. Choi presented two posters at international conferences on the work she has done. Furthermore, Dr. Choi completed training for animal handling and has expressed interest to train undergraduate students, which will aid in the development of her as an independent scientist. How have the results been disseminated to communities of interest?Inter-group presentations were arranged amonglab groups within our deparmtent, and via online meetings with our collaborators in Ireland and the UK. Audience members includedgraduate students, academic staff and faculty in the participating labs. The experiments conducted funded by this award yielded in this reporting period three publications, bothconference abstracts. These results were also presented in our lab meetings, and in on-campus seminar series. Dr. van Pijkeren was invited asan expert panelist by the NIH on the topic probiotics and engineered probiotics, a forum that attracted nearly 1,000 participants. What do you plan to do during the next reporting period to accomplish the goals?- Develop pull-down assay to determine which cell-wall-binding domain yields maximum binding activity; - Analyze existing RNAseq data to identify the strongest promoters for use of recombinant protein expression; - Produce array of chimeric lysins produced by probiotic LR; - Optimize the release of chimeric lysins from probiotic LR; - Obtain a large library of S. suis isolates that can be tested for susceptibility against recombinant lysin
Impacts
What was accomplished under these goals?
1. The probiotic L. reuteri was engineered to release a recombinant phage lysin; 2. As a proof-of-concept, we demonstrated the lysin is biologically active as we see modest antimicrobial activity against select S. suis strains; 3. To optimize protein production, high-throughput quantification is required. To this end, we developed a luminescent tagging system that allows in vitro, in vivo and in situ detection of recombinant bacteria and recombinant proteins.A manuscript is in preparation; 4. We initiated experiments to make chimeric lysins where different combinations of cell-wall binding domaind and lytic domains are fused; as a first step, we will determine optimized cell-wall binding, which will be accomplished by implementing our newly developed luminescent tagging system for pull-down assays.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
In-Young Choi, Jee-Hwan Oh and JP van Pijkeren. A luminescent tagging system to detect and quantify recombinant lactobacilli and their recombinant proteins. Beneficial Microbes Conference, Madison, WI, 2022
- Type:
Conference Papers and Presentations
Status:
Under Review
Year Published:
2022
Citation:
In-Young Choi, Jee-Hwan Oh and JP van Pijkeren. From Food to Gut: a Novel Luminescent Tagging System To Track Lactic Acid Bacteria. Korean Society of Food Science and Technology, Korea, 2022
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