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/).
The Housemaid was nominated for the Palme d'Or at the 2010 Cannes Film Festival. Critics praised Jeon Do-yeon’s haunting performance as Eun-yi and the film's ability to modernize a classic story while keeping the tension high. Where to Watch
The conflict intensifies as the women of the family—Hae-ra and her ruthless mother, Mi-hee—conspire to protect their social status and inheritance at all costs. Their actions, which include poisoning Eun-yi and forcing an abortion, highlight a world where empathy is traded for preservation of power.
: Provide background on "The Housemaid," discussing its original release, director, and any notable awards or controversies.
A biting social commentary on how the ultra-wealthy exploit the working class and use their power to maintain their status. Main Cast & Characters
Broken and vengeful, Eun-yi sneaks into the family’s Christmas party, hangs herself from the chandelier, and dies in front of the entire family and their guests — leaving them traumatized forever.
Ideal for mobile viewing and users with limited data or slower internet speeds.
When searching for this film online, always ensure you are using legitimate streaming platforms to avoid security risks. Many international films are now appearing on major services like Netflix, Amazon Prime, or specialized world cinema platforms.
The Housemaid was nominated for the Palme d'Or at the 2010 Cannes Film Festival. Critics praised Jeon Do-yeon’s haunting performance as Eun-yi and the film's ability to modernize a classic story while keeping the tension high. Where to Watch
The conflict intensifies as the women of the family—Hae-ra and her ruthless mother, Mi-hee—conspire to protect their social status and inheritance at all costs. Their actions, which include poisoning Eun-yi and forcing an abortion, highlight a world where empathy is traded for preservation of power.
: Provide background on "The Housemaid," discussing its original release, director, and any notable awards or controversies.
A biting social commentary on how the ultra-wealthy exploit the working class and use their power to maintain their status. Main Cast & Characters
Broken and vengeful, Eun-yi sneaks into the family’s Christmas party, hangs herself from the chandelier, and dies in front of the entire family and their guests — leaving them traumatized forever.
Ideal for mobile viewing and users with limited data or slower internet speeds.
When searching for this film online, always ensure you are using legitimate streaming platforms to avoid security risks. Many international films are now appearing on major services like Netflix, Amazon Prime, or specialized world cinema platforms.