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/).
Entrar al Jardín de Sangre y Huesos es pactar con un ecosistema hostil. No se puede domesticar a un león, y en Palo Mayombe, no se domestica a los muertos. Se les negocia, se les alimenta y se les ata, pero ellos siempre buscan la libertad.
Este artículo tiene fines culturales y antropológicos. Manipular huesos humanos sin el respeto, la cadena de linaje (la Rama ) y la preparación adecuada es peligroso mental y espiritualmente. Palo Mayombe es una religión cerrada; su llave es la iniciación, no la curiosidad mórbida. Si escuchas un perro ladrar a las tres de la mañana y el viento trae olor a tabaco y hierro, no mires hacia atrás. El Jardín te está observando a ti.
In Palo Mayombe, bones and blood are considered essential components of the spiritual practice. Bones are believed to hold the memories and energies of the ancestors and the spirits, while blood is seen as a symbol of life and vitality. The use of bones and blood in rituals and offerings is believed to connect the practitioner with the spiritual realm and to facilitate communication with the divine.
: Collected from specific trees to tap into the "spirit of the forest".
The work is noted for its "dignified" treatment of a much-maligned subject. Rather than focusing on lurid myths, it presents Palo as a serious system of spiritual integrity. For Practitioners: It serves as a guide for those seeking initiation or active , offering access to rare materials and field notes. For Scholars: Reviewers from
Palo Mayombe: El Jardín de Sangre y Huesos Palo Mayombe, often referred to as "the garden of blood and bones" ( el jardín de sangre y huesos ), is a powerful and frequently misunderstood Afro-Cuban religion. Rooted in the ancestral traditions of the Congo Basin in Central Africa, it was carried across the Atlantic during the slave trade and flourished in Cuba as a "New World Creole" religion.
Why is it called a Garden of Blood? In Palo, energy is not abstract; it is tangible and transferable. The concept of Nsala (vital force) is paramount. Just as a biological garden requires water and fertilizer to grow, the spiritual garden of the Nganga requires nourishment.
When enslaved Africans were brought to Cuba, they merged their ancestral beliefs with elements of Catholicism and Spiritism to survive the colonial environment. Today, it exists in several branches, with the main ones being , Briyumba , Monte , and Kimbisa . The Central Mystery: The Nganga
It provides detailed descriptions of the "tools" of the trade, including: Divination methods and sacred songs/chants. The use of herbs, animals, and minerals in ritual work.
The word translates to "stick" in Spanish, referencing the sacred wooden branches (palos) that are central to its rituals. The religion, also known as Regla de Congo or Palo Monte , draws heavily from traditional Kongo cosmology.
La marca en la espalda, los cortes rituales en el cuero cabelludo (acta de defunción espiritual) y el juramento de silencio sellan el pacto. A partir de ese momento, el iniciado no solo cuida el Jardín... es parte del Jardín .
Entrar al Jardín de Sangre y Huesos es pactar con un ecosistema hostil. No se puede domesticar a un león, y en Palo Mayombe, no se domestica a los muertos. Se les negocia, se les alimenta y se les ata, pero ellos siempre buscan la libertad.
Este artículo tiene fines culturales y antropológicos. Manipular huesos humanos sin el respeto, la cadena de linaje (la Rama ) y la preparación adecuada es peligroso mental y espiritualmente. Palo Mayombe es una religión cerrada; su llave es la iniciación, no la curiosidad mórbida. Si escuchas un perro ladrar a las tres de la mañana y el viento trae olor a tabaco y hierro, no mires hacia atrás. El Jardín te está observando a ti.
In Palo Mayombe, bones and blood are considered essential components of the spiritual practice. Bones are believed to hold the memories and energies of the ancestors and the spirits, while blood is seen as a symbol of life and vitality. The use of bones and blood in rituals and offerings is believed to connect the practitioner with the spiritual realm and to facilitate communication with the divine. Palo Mayombe- El Jardin de Sangre y Huesos
: Collected from specific trees to tap into the "spirit of the forest".
The work is noted for its "dignified" treatment of a much-maligned subject. Rather than focusing on lurid myths, it presents Palo as a serious system of spiritual integrity. For Practitioners: It serves as a guide for those seeking initiation or active , offering access to rare materials and field notes. For Scholars: Reviewers from Entrar al Jardín de Sangre y Huesos es
Palo Mayombe: El Jardín de Sangre y Huesos Palo Mayombe, often referred to as "the garden of blood and bones" ( el jardín de sangre y huesos ), is a powerful and frequently misunderstood Afro-Cuban religion. Rooted in the ancestral traditions of the Congo Basin in Central Africa, it was carried across the Atlantic during the slave trade and flourished in Cuba as a "New World Creole" religion.
Why is it called a Garden of Blood? In Palo, energy is not abstract; it is tangible and transferable. The concept of Nsala (vital force) is paramount. Just as a biological garden requires water and fertilizer to grow, the spiritual garden of the Nganga requires nourishment. Este artículo tiene fines culturales y antropológicos
When enslaved Africans were brought to Cuba, they merged their ancestral beliefs with elements of Catholicism and Spiritism to survive the colonial environment. Today, it exists in several branches, with the main ones being , Briyumba , Monte , and Kimbisa . The Central Mystery: The Nganga
It provides detailed descriptions of the "tools" of the trade, including: Divination methods and sacred songs/chants. The use of herbs, animals, and minerals in ritual work.
The word translates to "stick" in Spanish, referencing the sacred wooden branches (palos) that are central to its rituals. The religion, also known as Regla de Congo or Palo Monte , draws heavily from traditional Kongo cosmology.
La marca en la espalda, los cortes rituales en el cuero cabelludo (acta de defunción espiritual) y el juramento de silencio sellan el pacto. A partir de ese momento, el iniciado no solo cuida el Jardín... es parte del Jardín .