[P008] Poster

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

[P04-550] Optimization and Application of Methods for Functional Elucidation of Hypothetical Proteins in Bacteria Using Cell-Penetrating Peptides

○Tomoka Fujioka¹, Takahiro Muraoka¹, Tetsushi Mori¹ (1. Tokyo University of Agriculture and Technology (Japan))

Keywords:

Cell-Penetrating Peptides (CPP),Peptide Nucleic Acid(PNA),functional elucidation,Hypothetical Protein

Conventional intracellular biomolecule delivery has relied heavily on electroporation, conjugation, and transduction. However, these methods face significant limitations including low transformation efficiency and restricted applicability to novel or hard-to-transform bacterial species. To overcome these constraints, we investigated cell-penetrating peptides (CPPs) as an alternative delivery platform. CPPs are short peptides containing fewer than 40 amino acid residues that naturally permeate cellular membranes, positioning them as ideal carriers for biomolecules including proteins and nucleic acids. To optimize CPP efficiency across diverse bacterial strains, we systematically evaluated structural and abiotic factors affecting membrane permeation. Using Escherichia coli and other members of the Enterobacteriaceae family, we discovered that temperature and solution tonicity significantly influenced CPP permeation efficiency. From a structural perspective, we successfully enhanced CPP permeation by strategically modifying the side chain length of constituent amino acid residues. We subsequently applied this optimized approach to elucidate protein function in vivo within Paenibacillus, a bacterium difficult to transform using conventional methods. Initially, we evaluated the capacity of CPP-peptide nucleic acid (CPP-PNA) conjugates to regulate protein translation in the target organism. A specific probe targeting the essential acpP gene was synthesized and introduced into the culture medium. Growth inhibition was observed after 24 hours when CPP-PNA was administered at concentrations of 6 μM or higher. Importantly, cell growth recovery was confirmed after 36 hours, demonstrating that CPP-PNA treatment was non-toxic to the target bacterium. Subsequently, specialized CPP-PNA probes were designed and applied to target candidate genes cgiA and cgiB, which are predicted to encode ι-carrageenase enzymes. Enzymatic degradation of carrageenan substrate was evaluated based on viscosity reduction measurements, with successful degradation confirmed by observing increased liquid surface level during stirring as solution viscosity decreased. When probes targeting cgiB were applied during carrageenan degradation assays, no significant viscosity decrease was observed in the culture medium. To understand these experimental results, we compared the tertiary structures of cgiA and cgiB proteins with established ι-carrageenase structures to determine whether they possessed carrageenase-specific structural features. Results confirmed that both candidate genes lacked Domain A, which is specifically involved in ι-carrageenan degradation processes. However, comparative analysis of secondary structures revealed high similarity to other characterized ι-carrageenases, and all catalytic sites related to carrageenan degradation were highly conserved across all examined proteins. These comprehensive findings successfully confirmed the biological function of candidate carrageenase-encoding genes within the bacterial system. In summary, CPPs represent a powerful and versatile alternative approach for efficiently delivering biomolecules into diverse bacterial species, enabling comprehensive functional analyses to be performed in vivo without the limitations associated with conventional transformation methodologies.