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/).
These platforms typically host "lite" or censored previews, with descriptions directing users to the full versions. Content Safety Note
: Fans frequently cite his animations for their smooth 3D rendering and attention to detail, which was considered advanced for the time. DeviantArt Finding "Full" Content
For those looking for "full" content, users often suggest following their official social handles or visiting community-hosted galleries where long-form animations are archived. Giantess Jclain
: Jcalin Giantess Full could refer to a character or a specific storyline within a fictional work, possibly in adult literature, manga, or another form of media that features giantess fantasies.
“Jcalin” might be the name of a giantess character in a specific comic or game. For instance, user-created content in RPG Maker games (e.g., Mega Neko , Giantess Saya ) often features original named giants. “Full” would then refer to the completed game or uncensored art pack.
The artist is recognized for smooth 3D rendering and attention to architectural scale. Where to Find Full Content
and potentially through subscription services like Patreon, though many of his older official channels have become inactive or difficult to access.
These platforms typically host "lite" or censored previews, with descriptions directing users to the full versions. Content Safety Note
: Fans frequently cite his animations for their smooth 3D rendering and attention to detail, which was considered advanced for the time. DeviantArt Finding "Full" Content
For those looking for "full" content, users often suggest following their official social handles or visiting community-hosted galleries where long-form animations are archived. Giantess Jclain
: Jcalin Giantess Full could refer to a character or a specific storyline within a fictional work, possibly in adult literature, manga, or another form of media that features giantess fantasies.
“Jcalin” might be the name of a giantess character in a specific comic or game. For instance, user-created content in RPG Maker games (e.g., Mega Neko , Giantess Saya ) often features original named giants. “Full” would then refer to the completed game or uncensored art pack.
The artist is recognized for smooth 3D rendering and attention to architectural scale. Where to Find Full Content
and potentially through subscription services like Patreon, though many of his older official channels have become inactive or difficult to access.