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/).
Food is perhaps the most delicious entry point into Indian culture. Indian cuisine is a reflection of the land’s diversity—varying drastically from the coconut-infused curries of the South to the robust, wheat-based breads of the North. However, the Indian lifestyle regarding food goes beyond mere taste; it is rooted in the science of Ayurveda , emphasizing the balance of the body's constitution. The traditional Thali —a platter offering a variety of flavors (sweet, sour, salty, spicy, bitter, and astringent)—exemplifies the holistic approach to a meal. Furthermore, the concept of Atithi Devo Bhava ("the guest is equivalent to God") ensures that hospitality is a cornerstone of daily life, where feeding a guest is considered a moral duty rather than a mere social obligation.
Lifestyle in India is fundamentally communal. Unlike the individualistic cultures of the West, Indians rarely eat alone or celebrate alone.
Eating is considered a sacred act. In many traditional homes, sitting on the floor and eating with the right hand is still practiced to foster a connection with the food. 4. Spiritual Wellness and Mindful Living
Forget the cold brew and the frantic scroll through Instagram. The traditional Indian dinacharya (daily routine) is having a major comeback—with a Gen Z twist.
At the heart of Indian lifestyle lies the joint family system, though its prevalence is evolving. Traditionally, multiple generations—grandparents, parents, uncles, aunts, and cousins—lived under one roof, sharing resources, responsibilities, and a collective identity. This structure provides a robust social safety net, care for the elderly, and a built-in support system for child-rearing. However, urbanization and economic pressures have led to a rise in nuclear families, especially in metropolitan cities like Mumbai, Delhi, and Bengaluru. Yet, even in nuclear setups, familial ties remain extraordinarily strong, with frequent visits, phone calls, and financial interdependence binding members across distances.
Food is perhaps the most delicious entry point into Indian culture. Indian cuisine is a reflection of the land’s diversity—varying drastically from the coconut-infused curries of the South to the robust, wheat-based breads of the North. However, the Indian lifestyle regarding food goes beyond mere taste; it is rooted in the science of Ayurveda , emphasizing the balance of the body's constitution. The traditional Thali —a platter offering a variety of flavors (sweet, sour, salty, spicy, bitter, and astringent)—exemplifies the holistic approach to a meal. Furthermore, the concept of Atithi Devo Bhava ("the guest is equivalent to God") ensures that hospitality is a cornerstone of daily life, where feeding a guest is considered a moral duty rather than a mere social obligation.
Lifestyle in India is fundamentally communal. Unlike the individualistic cultures of the West, Indians rarely eat alone or celebrate alone.
Eating is considered a sacred act. In many traditional homes, sitting on the floor and eating with the right hand is still practiced to foster a connection with the food. 4. Spiritual Wellness and Mindful Living
Forget the cold brew and the frantic scroll through Instagram. The traditional Indian dinacharya (daily routine) is having a major comeback—with a Gen Z twist.
At the heart of Indian lifestyle lies the joint family system, though its prevalence is evolving. Traditionally, multiple generations—grandparents, parents, uncles, aunts, and cousins—lived under one roof, sharing resources, responsibilities, and a collective identity. This structure provides a robust social safety net, care for the elderly, and a built-in support system for child-rearing. However, urbanization and economic pressures have led to a rise in nuclear families, especially in metropolitan cities like Mumbai, Delhi, and Bengaluru. Yet, even in nuclear setups, familial ties remain extraordinarily strong, with frequent visits, phone calls, and financial interdependence binding members across distances.