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
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For fans of the genre, the scene represents the gold standard of the "Raw" format: high-end talent, exceptional visual quality, and a performance that feels genuinely energetic rather than choreographed.
The longevity of "-BlackedRaw- Liya Silver - Joy Ride -" can be attributed to the "lightning in a bottle" chemistry captured during this period of Liya's career. In early 2019, she was transitioning from a rising star to a top-tier performer, and this scene captured that peak energy.
Years after its release, this specific date and title are frequently cited by fans as a "classic" Liya Silver scene. It helped solidify her status within the Blacked network, eventually leading to her winning multiple industry awards in the years following. Why It Remains Popular
By 2019, Liya Silver had already established herself as a powerhouse in the industry. With her signature blonde hair, slender athletic build, and expressive performances, the Russian-born starlet became a favorite for directors seeking a blend of elegance and intensity. "Joy Ride" served as a perfect showcase for her versatility, moving away from stylized sets and focusing on the chemistry between the performers. The BlackedRaw Aesthetic
For fans of the genre, the scene represents the gold standard of the "Raw" format: high-end talent, exceptional visual quality, and a performance that feels genuinely energetic rather than choreographed.
The longevity of "-BlackedRaw- Liya Silver - Joy Ride -" can be attributed to the "lightning in a bottle" chemistry captured during this period of Liya's career. In early 2019, she was transitioning from a rising star to a top-tier performer, and this scene captured that peak energy.
Years after its release, this specific date and title are frequently cited by fans as a "classic" Liya Silver scene. It helped solidify her status within the Blacked network, eventually leading to her winning multiple industry awards in the years following. Why It Remains Popular
By 2019, Liya Silver had already established herself as a powerhouse in the industry. With her signature blonde hair, slender athletic build, and expressive performances, the Russian-born starlet became a favorite for directors seeking a blend of elegance and intensity. "Joy Ride" served as a perfect showcase for her versatility, moving away from stylized sets and focusing on the chemistry between the performers. The BlackedRaw Aesthetic