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Published on: February 22, 2017
Investigation of Intracellular Clearing of Streptococcus pneumoniae by mRNA-Encoded Cpl-1 Bacteriophage Endolysin in
Carolin Warnke1, Wendy Bergmann-Ewert2, Marc Benjamin Janssen1
1Institute of Medical Microbiology, Virology and Hygiene, University Medicine Rostock, 18057 Rostock, Germany.
Abstract:
Streptococcus pneumoniae remains a major global health threat and is listed by the World Health Organization as a pathogen in urgent need of new antimicrobial strategies. While primarily considered an extracellular pathogen, S. pneumoniae can persist within splenic macrophages in severe disease, creating a protected intracellular niche that may contribute to fulminant sepsis. We recently demonstrated the concept of an mRNA-based therapeutic approach in which host cells produce the pneumococcal bacteriophage endolysin Cpl-1. Here, we investigated whether expression of Cpl-1 in macrophages can target S. pneumoniae residing within host cells. Using the human THP-1 macrophage line, we demonstrated successful translation and intracellular accumulation of bioactive Cpl-1 following IVT-mRNA transfection. Lysates from Cpl-1 mRNA-transfected cells exhibited bacteriolytic activity, and Western blotting as well as immunofluorescent staining confirmed cytosolic endolysin production. Phagocytosis assays using an encapsulated and unencapsulated pneumococcal strain showed a reduction in intracellular bacterial burden in Cpl-1 mRNA-transfected macrophages compared with control and inactive-mutant Cpl-1 mRNA groups, and a flow cytometry-based assay further corroborated a decreased intracellular bacterial signal. Together, these findings suggest that mRNA-encoded Cpl-1 enhances intracellular killing of S. pneumoniae and supports the feasibility of mRNA-based endolysin therapies to target intracellular pneumococcal reservoirs.
Insights
Messenger RNA (mRNA) therapy can equip macrophages to produce Cpl-1, an enzyme that kills intracellular Streptococcus pneumoniae. This approach shows promise for combating pneumococcal infections by targeting bacteria within host cells.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Streptococcus pneumoniae is a major global health threat requiring novel antimicrobial strategies.
- Intracellular persistence of S. pneumoniae in macrophages can lead to severe sepsis.
- Previous work established mRNA therapeutics for host cell production of pneumococcal endolysin Cpl-1.
Purpose of the Study:
- To investigate if Cpl-1 expression in macrophages can target intracellular S. pneumoniae.
- To evaluate the efficacy of mRNA-encoded Cpl-1 against intracellular pneumococcal infections.
Main Methods:
- Transfection of human THP-1 macrophages with in vitro transcribed mRNA encoding Cpl-1.
- Assessing Cpl-1 bioactivity and intracellular accumulation via Western blotting and immunofluorescence.
- Quantifying intracellular bacterial burden using phagocytosis assays and flow cytometry.
Main Results:
- Successful translation and accumulation of bioactive Cpl-1 within macrophages.
- Cpl-1 mRNA-transfected cells demonstrated bacteriolytic activity against S. pneumoniae.
- Significant reduction in intracellular S. pneumoniae burden observed in Cpl-1-treated macrophages.
Conclusions:
- mRNA-encoded Cpl-1 effectively enhances intracellular killing of S. pneumoniae.
- This strategy shows feasibility for targeting intracellular pneumococcal reservoirs.
- Supports the development of mRNA-based endolysin therapies for bacterial infections.
