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/).
Inclusion of accurate English subtitles ensures that viewers never miss a beat of Locke's fast-paced, sharp-witted dialogue or the subtle, muttered nuances of Eastwood's iconic gravelly voice amid the roaring engines and deafening gunfire. Final Thoughts
However, the routine assignment quickly turns into a nightmare. Gus Mally—played brilliantly by Sondra Locke, Eastwood’s frequent real-life and on-screen partner—is not a minor witness. She is an articulate, sharp-tongued, college-educated sex worker who possesses incriminating evidence involving high-ranking mobsters and corrupt police officials.
Two sequences in the film have achieved legendary status among action cinema aficionados:
Sondra Locke’s Gus Mally is the true catalyst of the narrative. Rather than playing a helpless damsel in distress, Mally is highly intelligent, defiant, and cynical of authority. She possesses a far better understanding of the criminal underworld and police corruption than Shockley does.
The Gauntlet (1977) stands as a pivotal moment in Clint Eastwood's illustrious career. Released during a time when Eastwood was cementing his status not just as a leading man, but as a formidable director, this film showcases a blend of high-stakes action, wry humor, and the gritty, character-driven narrative typical of 1970s cinema. For fans looking to revisit this classic in , the movie offers a thrilling ride through the Nevada and Arizona deserts, featuring iconic, chaotic stunt work.
Initially, Shockley treats Mally with casual condescension, viewing her through a lens of societal prejudice. However, Mally is frequently the smartest person in the room. She continuously saves Shockley from his own naivety, figuring out the ambushes before he does. As they rely on each other to survive, their mutual hostility softens into a deep, protective bond. Locke gives a fiery, fierce performance that holds its own against Eastwood's stoic screen presence, making their banter just as engaging as the shootouts. Why English Subtitles Matter for Modern Audiences
Inclusion of accurate English subtitles ensures that viewers never miss a beat of Locke's fast-paced, sharp-witted dialogue or the subtle, muttered nuances of Eastwood's iconic gravelly voice amid the roaring engines and deafening gunfire. Final Thoughts
However, the routine assignment quickly turns into a nightmare. Gus Mally—played brilliantly by Sondra Locke, Eastwood’s frequent real-life and on-screen partner—is not a minor witness. She is an articulate, sharp-tongued, college-educated sex worker who possesses incriminating evidence involving high-ranking mobsters and corrupt police officials.
Two sequences in the film have achieved legendary status among action cinema aficionados:
Sondra Locke’s Gus Mally is the true catalyst of the narrative. Rather than playing a helpless damsel in distress, Mally is highly intelligent, defiant, and cynical of authority. She possesses a far better understanding of the criminal underworld and police corruption than Shockley does.
The Gauntlet (1977) stands as a pivotal moment in Clint Eastwood's illustrious career. Released during a time when Eastwood was cementing his status not just as a leading man, but as a formidable director, this film showcases a blend of high-stakes action, wry humor, and the gritty, character-driven narrative typical of 1970s cinema. For fans looking to revisit this classic in , the movie offers a thrilling ride through the Nevada and Arizona deserts, featuring iconic, chaotic stunt work.
Initially, Shockley treats Mally with casual condescension, viewing her through a lens of societal prejudice. However, Mally is frequently the smartest person in the room. She continuously saves Shockley from his own naivety, figuring out the ambushes before he does. As they rely on each other to survive, their mutual hostility softens into a deep, protective bond. Locke gives a fiery, fierce performance that holds its own against Eastwood's stoic screen presence, making their banter just as engaging as the shootouts. Why English Subtitles Matter for Modern Audiences