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/).
To help me write the most relevant article for you, could you please share: general report on the site? What is the target audience for this article (e.g., film students, general viewers)? legal alternatives I can then provide a more tailored and detailed draft. Monzo - Mobile Banking - Apps on Google Play
This public link is valid for 7 days and shares a thread, including any personal information you added. This link or copies made by others cannot be deleted. If you share with third parties, their policies apply. Can’t copy the link right now. Try again later.
[User Request] ──> [Proxy/Mirror Domain] ──> [Ad-Network Overlay] ──> [Offshore File Host] 1. Domain Hopping and Mirror Links
The global leader in streaming. While it is a paid subscription service, the quality of content is unmatched. They invest heavily in Indian cinema, producing and acquiring rights to major films.
Sites like Afimywap utilize specific digital tactics to remain online and monetize traffic:
. While the site is a common destination for free streaming and downloads, it is important to understand its impact on the film industry and the legal risks involved for users. 🎬 Overview of Afilmywap
The primary victim of these platforms is the creative industry itself. Piracy is estimated to cost the global film industry billions of dollars annually in lost revenue. In India, laws like the strictly prohibit the distribution of copyrighted material without authorization. When users choose sites like Afilmywap over official channels, it directly impacts the box office performance and the ability of creators to fund future projects. Risks to the Consumer
While the legal risks are significant, the immediate danger for the average user is cybersecurity. There have been verified reports of similar piracy websites being hijacked by hackers with the sole purpose of tricking visitors into installing malware on their devices.
: Regional language content catering to specific North Indian demographics.
: It is particularly popular for providing dubbed versions of Hollywood films and regional Indian cinema (e.g., Hindi, Punjabi, and South Indian movies).
To help me write the most relevant article for you, could you please share: general report on the site? What is the target audience for this article (e.g., film students, general viewers)? legal alternatives I can then provide a more tailored and detailed draft. Monzo - Mobile Banking - Apps on Google Play
This public link is valid for 7 days and shares a thread, including any personal information you added. This link or copies made by others cannot be deleted. If you share with third parties, their policies apply. Can’t copy the link right now. Try again later.
[User Request] ──> [Proxy/Mirror Domain] ──> [Ad-Network Overlay] ──> [Offshore File Host] 1. Domain Hopping and Mirror Links
The global leader in streaming. While it is a paid subscription service, the quality of content is unmatched. They invest heavily in Indian cinema, producing and acquiring rights to major films.
Sites like Afimywap utilize specific digital tactics to remain online and monetize traffic:
. While the site is a common destination for free streaming and downloads, it is important to understand its impact on the film industry and the legal risks involved for users. 🎬 Overview of Afilmywap
The primary victim of these platforms is the creative industry itself. Piracy is estimated to cost the global film industry billions of dollars annually in lost revenue. In India, laws like the strictly prohibit the distribution of copyrighted material without authorization. When users choose sites like Afilmywap over official channels, it directly impacts the box office performance and the ability of creators to fund future projects. Risks to the Consumer
While the legal risks are significant, the immediate danger for the average user is cybersecurity. There have been verified reports of similar piracy websites being hijacked by hackers with the sole purpose of tricking visitors into installing malware on their devices.
: Regional language content catering to specific North Indian demographics.
: It is particularly popular for providing dubbed versions of Hollywood films and regional Indian cinema (e.g., Hindi, Punjabi, and South Indian movies).