Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing Reagents
Phage endolysin-specific binding characteristics and killing activity support their potential use in biotechnological applications, including potency and purity testing of live biotherapeutic products (LBPs). LBPs contain live organisms, such as lactic acid bacteria (LAB), and are intended for use a...
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| Format: | Article |
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MDPI AG
2023-09-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/15/10/1986 |
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| author | Robert J. Dorosky Stephanie L. Lola Haleigh A. Brown Jeremy E. Schreier Sheila M. Dreher-Lesnick Scott Stibitz |
| author_facet | Robert J. Dorosky Stephanie L. Lola Haleigh A. Brown Jeremy E. Schreier Sheila M. Dreher-Lesnick Scott Stibitz |
| author_sort | Robert J. Dorosky |
| collection | DOAJ |
| description | Phage endolysin-specific binding characteristics and killing activity support their potential use in biotechnological applications, including potency and purity testing of live biotherapeutic products (LBPs). LBPs contain live organisms, such as lactic acid bacteria (LAB), and are intended for use as drugs. Our approach uses the endolysin cell wall binding domains (CBD) for LBP potency assays and the endolysin killing activity for purity assays. CBDs of the following five lactobacilli phage lysins were characterized: CL1, Jlb1, Lj965, LL-H, and ΦJB. They exhibited different bindings to 27 LAB strains and were found to bind peptidoglycan or surface polymers. Flow cytometry based on CBD binding was used to enumerate viable counts of two strains in the mixture. CL1-lys, jlb1-lys, and ΦJB-lys and their enzymatic domains (EADs) exhibited cell wall digestive activity and lytic activity against LAB. Jlb1-EAD and ΦJB-EAD were more sensitive than their respective hololysins to buffer pH and NaCl changes. The ΦJB-EAD exhibited stronger lytic activity than ΦJB-lys, possibly due to ΦJB-CBD-mediated sequestration of ΦJB-lys by cell debris. CBD multiplex assays indicate that these proteins may be useful LBP potency reagents, and the lytic activity suggests that CL1-lys, jlb1-lys, and ΦJB-lys and their EADs are good candidates for LBP purity reagent development. |
| first_indexed | 2024-03-10T20:49:44Z |
| format | Article |
| id | doaj.art-4112c26b114446dd9b123ac1a44dd1e2 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T20:49:44Z |
| publishDate | 2023-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-4112c26b114446dd9b123ac1a44dd1e22023-11-19T18:26:29ZengMDPI AGViruses1999-49152023-09-011510198610.3390/v15101986Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing ReagentsRobert J. Dorosky0Stephanie L. Lola1Haleigh A. Brown2Jeremy E. Schreier3Sheila M. Dreher-Lesnick4Scott Stibitz5Office of Vaccines Research and Review, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USAOffice of Vaccines Research and Review, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USAOffice of Vaccines Research and Review, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USADepartment of Marine Sciences, University of Georgia, Athens, GA 30602, USAOffice of Vaccines Research and Review, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USAOffice of Vaccines Research and Review, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USAPhage endolysin-specific binding characteristics and killing activity support their potential use in biotechnological applications, including potency and purity testing of live biotherapeutic products (LBPs). LBPs contain live organisms, such as lactic acid bacteria (LAB), and are intended for use as drugs. Our approach uses the endolysin cell wall binding domains (CBD) for LBP potency assays and the endolysin killing activity for purity assays. CBDs of the following five lactobacilli phage lysins were characterized: CL1, Jlb1, Lj965, LL-H, and ΦJB. They exhibited different bindings to 27 LAB strains and were found to bind peptidoglycan or surface polymers. Flow cytometry based on CBD binding was used to enumerate viable counts of two strains in the mixture. CL1-lys, jlb1-lys, and ΦJB-lys and their enzymatic domains (EADs) exhibited cell wall digestive activity and lytic activity against LAB. Jlb1-EAD and ΦJB-EAD were more sensitive than their respective hololysins to buffer pH and NaCl changes. The ΦJB-EAD exhibited stronger lytic activity than ΦJB-lys, possibly due to ΦJB-CBD-mediated sequestration of ΦJB-lys by cell debris. CBD multiplex assays indicate that these proteins may be useful LBP potency reagents, and the lytic activity suggests that CL1-lys, jlb1-lys, and ΦJB-lys and their EADs are good candidates for LBP purity reagent development.https://www.mdpi.com/1999-4915/15/10/1986phage endolysinphage lysincell wall binding domainenzymatic activity domainmicrobial purity<i>Lactobacillus</i> |
| spellingShingle | Robert J. Dorosky Stephanie L. Lola Haleigh A. Brown Jeremy E. Schreier Sheila M. Dreher-Lesnick Scott Stibitz Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing Reagents Viruses phage endolysin phage lysin cell wall binding domain enzymatic activity domain microbial purity <i>Lactobacillus</i> |
| title | Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing Reagents |
| title_full | Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing Reagents |
| title_fullStr | Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing Reagents |
| title_full_unstemmed | Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing Reagents |
| title_short | Characterization of Lactobacilli Phage Endolysins and Their Functional Domains–Potential Live Biotherapeutic Testing Reagents |
| title_sort | characterization of lactobacilli phage endolysins and their functional domains potential live biotherapeutic testing reagents |
| topic | phage endolysin phage lysin cell wall binding domain enzymatic activity domain microbial purity <i>Lactobacillus</i> |
| url | https://www.mdpi.com/1999-4915/15/10/1986 |
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