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/).
: Identify official emergency anchorages (e.g., Eastern Clean Ore Anchorage or Western Holding Anchorage) prior to transit. Never anchor inside the TSS.
: The Master should take the conn well before entering the Singapore Strait TSS.
: Between specific coordinates in the westbound lanes of the Singapore Strait, vessels should not exceed 12 knots over ground whenever safe and practicable. Manoeuvring Readiness
While the keyword remains "pdf" due to offline reliability and ease of annotation, the industry is moving toward integrated into ECDIS. However, until satellite connectivity is 100% guaranteed in the Malacca Cellular Dead Zones, a downloaded, verified PDF remains the standard for:
Engines must be fully ready for immediate maneuver. Steering gear should be placed in manual control well before entering congested chokepoints; rely on hand-steering rather than autopilot.
High risk of traffic convergence as vessels cross the TSS to enter or leave Singapore port limits. Horsburgh Lighthouse The eastern gateway of the Singapore Strait.
Passage planning for the Malacca and Singapore Straits cannot be a simple copy-and-paste exercise on the ECDIS. It demands a rigorous, dynamic approach that integrates precise UKC calculations, acute environmental awareness, flawless execution of COLREGs, and proactive bridge communication. By carefully charting every waypoint and maintaining an enhanced state of vigilance, bridge teams can navigate this vital global trade artery safely and efficiently. To advance your voyage planning,
The PPG contains a specific to the straits, including:
To help me tailor any specific details or sections of this guide for your needs, could you share a bit more information? What is the of your vessel?
Following the standard IMO framework, the passage plan must be broken down into four distinct phases: Appraisal, Planning, Execution, and Monitoring. Phase 1: Appraisal (Information Gathering)
: Identify official emergency anchorages (e.g., Eastern Clean Ore Anchorage or Western Holding Anchorage) prior to transit. Never anchor inside the TSS.
: The Master should take the conn well before entering the Singapore Strait TSS.
: Between specific coordinates in the westbound lanes of the Singapore Strait, vessels should not exceed 12 knots over ground whenever safe and practicable. Manoeuvring Readiness
While the keyword remains "pdf" due to offline reliability and ease of annotation, the industry is moving toward integrated into ECDIS. However, until satellite connectivity is 100% guaranteed in the Malacca Cellular Dead Zones, a downloaded, verified PDF remains the standard for:
Engines must be fully ready for immediate maneuver. Steering gear should be placed in manual control well before entering congested chokepoints; rely on hand-steering rather than autopilot.
High risk of traffic convergence as vessels cross the TSS to enter or leave Singapore port limits. Horsburgh Lighthouse The eastern gateway of the Singapore Strait.
Passage planning for the Malacca and Singapore Straits cannot be a simple copy-and-paste exercise on the ECDIS. It demands a rigorous, dynamic approach that integrates precise UKC calculations, acute environmental awareness, flawless execution of COLREGs, and proactive bridge communication. By carefully charting every waypoint and maintaining an enhanced state of vigilance, bridge teams can navigate this vital global trade artery safely and efficiently. To advance your voyage planning,
The PPG contains a specific to the straits, including:
To help me tailor any specific details or sections of this guide for your needs, could you share a bit more information? What is the of your vessel?
Following the standard IMO framework, the passage plan must be broken down into four distinct phases: Appraisal, Planning, Execution, and Monitoring. Phase 1: Appraisal (Information Gathering)