Innovations in Molecular Biotechnology

ABSTRACT

Advanced technologies have generated a wealth of microbial genomics data, resulting in numerous uncharacterized genes. A large portion of the Orientia tsutsugamushi proteome consists of such hypothetical and conserved proteins with poorly understood functions. In this study, we chose 292 hypothetical proteins from O. tsutsugamushi strains (Ikeda and Boryong) for systematic in-silico characterization. We used a series of computational tools (ExPASy ProtParam, VirulentPred, PSORTb, Pfam, InterProScan, PSIPRED, SWISS-MODEL, CASTp, and STRING) to predict physicochemical properties, subcellular localization, functional domains, secondary and tertiary structure, active sites, and protein–protein interactions. Comparative proteomic analysis identified stable proteins, cytoplasmic proteins, and virulent proteins. Notably, proteins that showed no significant homology to the human proteome but are likely key to bacterial function. Functional classification placed many hypothetical proteins at the enzyme class (e.g., phosphatases, transferases, hydrolases, kinases, peptidases, isomerases, ligases, oxidoreductases), transporters, binding proteins, secretory proteins, and regulatory factors. We generated homology models and validated models with multiple structural assessment metrics and identified areas of activity of interest for possible drug targeting.Our in-silico annotations provide hypotheses for the function of previously uncharacterized proteins of O. tsutsugamushi and provide a ranked set of prioritized targets for experimental laboratory validation and structure-based drug design.

KEYWORDS

1. Orientia tsutsugamushi
2. Hypothetical protein
3. In- silico analysis
4. Functional characterization