Characterization of Lactobacilli Phage Endolysins and Their Functional Domains-Potential Live Biotherapeutic Testing Reagents

Viruses. 2023 Sep 23;15(10):1986. doi: 10.3390/v15101986.

Abstract

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.

Keywords: Lactobacillus; cell wall binding domain; enzymatic activity domain; lactobacilli; microbial purity; phage endolysin; phage lysin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteriophages* / metabolism
  • Cell Wall / metabolism
  • Endopeptidases / metabolism
  • Lactobacillus
  • Peptidoglycan / metabolism

Substances

  • endolysin
  • Endopeptidases
  • Peptidoglycan

Grants and funding

This work was funded by an Interagency agreement between FDA/CBER and NIH/NIAID (AAI17016-001-00001). This project was supported in part by the Research Fellowship Program at the Office of Vaccines Research and Review/Center for Biologics Evaluation and Research, FDA, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and FDA.