Functional and Structure Prediction of Hypothetical Proteins From Listeria aquatica FSL S10-1188: Bioinformatics...

Listeria aquatica FSL S10-1188 is a freshwater bacterium characterized by distinctive metabolic properties and a genome enriched in hypothetical proteins (HPs). Although it is not considered pathogenic, its genomic features and aquatic habitat raise concerns about potential involvement in horizontal antimicrobial resistance (AMR) gene transfer.

The genome of L. aquatica FSL S10-1188 (GenBank Accession: CP011539.1) contains 2789 predicted proteins that were initially screened. From these, 919 HPs were prioritized based on sequence length (> 50 amino acids), physicochemical stability assessed using ProtParam, and consistent subcellular localization predictions from CELLO, PSORTb, and PSLpred. Among these, two stable cytoplasmic, HPs EUJ18943.1 and EUJ18676.1, were selected for comprehensive characterization. Functional insights were explored through BLASTp, secondary structure prediction, and 3D structural modeling using AlphaFold3. Model quality was validated using multiple tools including SAVES v6.0 and QMEANDisCo. Virulence potential was predicted via VICMpred, VirulentPred, and DeepVF. Putative ligand-binding sites were identified using PrankWeb, and molecular docking analyses were conducted using ProBiS for ligand prediction and AutoDock to evaluate ligand-binding affinity.

EUJ18943.1 (136 aa) and EUJ18676.1 (206 aa) are stable, hydrophilic proteins with acidic isoelectric points. BLASTp identified EUJ18943.1 as homologous to Imm48 immunity proteins and EUJ18676.1 as a GyrI-like detoxification protein. Both showed alpha-helix-rich secondary structures, with high-confidence AlphaFold3 models (pTM scores: 0.87 and 0.94). Structural validation confirmed the model quality. Virulence prediction tools classified both as potential virulence factors. Active sites were predicted by PrankWeb and ProBiS, identifying kanamycin A (score: 2.32) and streptomycin (score: 2.10) as top ligands. AutoDock v4.5.6 revealed strong binding affinities (Δ G = -7.17 and - 4.30 kcal/mol, respectively).

This study provides the first in silico structural and functional characterization of two HPs from L. aquatica FSL S10-1188, suggesting roles in stress response and virulence. These findings highlight their potential in AMR gene transfer and demonstrate the importance of computational annotation for guiding future experimental validation.