Aliarcobacter butzleri is an emerging foodborne and zoonotic pathogen, yet many of its encoded proteins remain functionally uncharacterized. This lack of annotation limits understanding of its molecular mechanisms and hampers the identification of novel therapeutic targets. In this study, we systematically performed functional annotation of essential hypothetical proteins from the BNI-3166 strain using an integrative-in-silico approach to uncover potential drug and vaccine candidates. 2,367 protein-coding sequences were retrieved from the RefSeq database and were identified 356 as hypothetical proteins. Using BLASTp, we screened these HPs against the Database of Essential Genes and the human proteome to identify essential non-homologous proteins, resulting in 20 ENH candidates. Functional annotation was performed using several domain-based databases, including Pfam, InterPro, SMART, and SUPERFAMILY. Subsequently, physicochemical properties were analyzed and predicted subcellular localization using PSORTb and CELLO. To assess druggability, the ChEMBL database was used. Virulence factors using VFDB, VICMpred, and VirulentPred 2.0 were also predicted. Gene Ontology annotations were generated via ARGOT2.5. Furthermore, we explored protein-protein interactions using STRING and predicted tertiary structures with AlphaFold3. Moreover, Ligand binding pockets were predicted using PrankWeb, and antigenicity of vaccine candidates was assessed using VaxiJen v2.0. We identified 20 essential non-homologous hypothetical proteins, of which 10 were confidently annotated based on conserved domain analysis. These proteins were classified as enzymes, binding proteins, transporters, regulatory proteins, and potential virulence factors. Among them, eight exhibited characteristics of promising drug targets, while two showed potential as vaccine candidates based on subcellular localization. Druggability analysis revealed that nine proteins had no similarity to known drug targets, suggesting novel therapeutic potential. Predicted 3D structures generated using AlphaFold3 yielded pTM scores ranging from 0.44 to 0.92, indicating acceptable to high modeling confidence. Ligand binding site analysis confirmed druggability in six candidates, and antigenicity screening identified one protein as a potential vaccine target. This study provides a computational framework for identifying functionally important proteins in A. butzleri BNI-3166 and highlights novel therapeutic candidates for experimental validation, offering new directions in drug and vaccine development against this underexplored pathogen.
Key words: Aliarcobacter butzleri, Drug Target Identification, Functional Annotation, Hypothetical Proteins, In Silico Analysis
Received: 08.07.2025; Accepted: 01.09.2025; Early view: 24.09.2025 Published: 10.01.2026
DOI: 10.62063/ecb-66
Citation: Paul, S., Barua, S., & Barua, J.D. (2026). In-silico functional annotation and structural characterization of hypothetical proteins from Aliarcobacter butzleri BNI-3166: Insights into novel virulence and drug targets. The European chemistry and biotechnology journal, 5, 22-39. https://doi.org/10.62063/ecb-66
The copyrights of the studies published in The European Chemistry and Biotechnology Journal (EUCHEMBIOJ) belong to their authors
This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).
Disney+ and HuluIn a shift from the first season, Tokyo Revengers Season 2 is primarily licensed by Disney. In the United States, episodes are available on Hulu, while international fans can find them on Disney+ under the "Star" banner. Both platforms allow premium subscribers to download episodes directly to mobile devices for offline viewing.
This season focuses on the conflict between Toman and the Black Dragon gang, currently led by the ruthless Taiju Shiba. The narrative centers on Hakkai Shiba’s struggle to protect his sister, Yuzuha, from their brother's violence. It is a season defined by snowy battles, the strength of brotherhood, and Takemichi’s evolving resolve to become the true "hero" of the story.
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Disney+ and HuluIn a shift from the first season, Tokyo Revengers Season 2 is primarily licensed by Disney. In the United States, episodes are available on Hulu, while international fans can find them on Disney+ under the "Star" banner. Both platforms allow premium subscribers to download episodes directly to mobile devices for offline viewing.
This season focuses on the conflict between Toman and the Black Dragon gang, currently led by the ruthless Taiju Shiba. The narrative centers on Hakkai Shiba’s struggle to protect his sister, Yuzuha, from their brother's violence. It is a season defined by snowy battles, the strength of brotherhood, and Takemichi’s evolving resolve to become the true "hero" of the story.
Tokyo Revengers Season 2 English Subtitles Download The return of Takemichi Hanagaki and the Tokyo Manji Gang has sparked a massive surge in fans looking to secure the latest episodes for offline viewing. Following the explosive "Bloody Halloween" arc, the second season—officially titled Tokyo Revengers: Christmas Showdown—delivers higher stakes, deeper betrayals, and the introduction of the formidable Shiba family. If you are looking to find Tokyo Revengers Season 2 with English subtitles for download, this guide covers everything you need to know about the plot, official sources, and file formats. The Plot: The Christmas Showdown Arc
Tokyo Revengers Season 2 is a masterclass in tension and character development. Whether you are watching Takemichi confront Taiju Shiba in the church or unraveling Kisaki’s latest scheme, having a high-quality download with accurate English subtitles is essential for the best experience. Stick to official platforms for the best security, or ensure your local media player is updated to handle the latest MKV softsubs. If you want to know more about the Tokyo Revengers series: Check out the manga chapters covered in Season 2. Look up the voice cast for the new characters. Explore the official soundtrack.