[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-556] Translation Inhibition and Spore-Selective Isolation against Gram-Positive Bacteria using Cell-Penetrating Peptides

○Kentaro Kurihara¹, Jaeyoung Yu¹, Takahiro Muraoka¹, Tetsushi Mori¹ (1. Tokyo University of Agriculture and Technology (Japan))

Keywords:

Cell-penetrating peptides,Gram-positive bacteria,Endospores

Gram-positive bacteria are abundant and highly diverse microorganisms that play crucial roles in environmental microbial communities, clinical settings, and industrial applications. They contribute significantly to nutrient cycling, symbiotic interactions, and bioactive compound production, while also including many important pathogenic species. Despite their ecological and biomedical significance, Gram-positive bacteria remain less understood than their Gram-negative counterparts, largely due to their thick, rigid peptidoglycan cell wall that creates a formidable barrier to molecular tool delivery. Additionally, certain physiological states such as spore formation further complicate molecular access and experimental manipulation. Conventional delivery methods, including electroporation and chemical transformation, often exhibit low efficiency or limited applicability across diverse species, thereby hindering functional studies and genetic manipulation efforts. To address these challenges, we explored cell-penetrating peptides (CPPs) as a non-genetic delivery platform. CPPs are short, cationic, amphipathic peptides capable of transporting diverse molecular cargoes across cellular membranes into cells. We systematically selected a panel of CPPs based on their physicochemical properties and conjugating them with carboxyfluorescein (FAM) to enable visualization and quantitative uptake evaluation. Delivery efficiency was assessed across multiple Gram-positive model strains using fluorescence microscopy and flow cytometry. Additionally, CPP–peptide nucleic acid (CPP–PNA) conjugates were employed to evaluate functional delivery capacity using antisense PNA sequences designed to inhibit protein translation of targeted genes. Our screening revealed that delivery efficiency varies significantly depending on peptide structure and target bacterial species. A Dab-rich CPP exhibited the highest delivery efficiency in vegetative cells, demonstrating strong intracellular fluorescence signals and broad cross-species compatibility. Flow cytometry analysis confirmed substantial increases in fluorescence-positive cell populations compared to untreated controls, indicating effective membrane translocation. Functional assays using CPP–PNA conjugates demonstrated dose-dependent growth inhibition patterns, confirming successful intracellular PNA delivery and subsequent translational inhibition. Interestingly, we identified an Orn-based peptide that showed selective permeability toward bacterial spores, enabling fluorescence labeling and sorting of spores from mixed cell populations. In summary, our findings demonstrate that CPPs provide a versatile and adaptable platform for addressing molecular delivery challenges in Gram-positive bacteria. The ability to achieve both broad-spectrum delivery in vegetative cells and selective spore targeting significantly expands system applicability. This approach enables intracellular delivery, targeted gene knockdown effects, and detection and manipulation of specific bacterial subpopulations. This work establishes the foundation for advanced applications in translational control, functional genomics, antimicrobial development, and environmental microbiology, contributing to deeper understanding of Gram-positive bacterial biology.