Identification and Characterization of Outer Membrane Proteins and Membrane Spanning Protein Complexes in Brucella melitensis

Abstract

Brucellosis (Malta fever) is a zoonotic disease that affects both humans and animals, including cattle, sheep, and goats. Brucella melitensis is the most virulent and clinically significant species in humans. It is a Gram-negative bacterium with three groups of outer membrane proteins (OMPs): minor OMPs (Group 1), and major OMPs (Groups 2 and 3). OMPs with {beta}-barrel architecture play important roles in nutrient transport, efflux, adhesion, and membrane biogenesis. Despite their importance, the structure, function, and interaction dynamics of several B. melitensis {beta}-barrel OMPs and associated protein complexes remain mostly unexplored. In this study, we conducted a comprehensive in silico analysis to characterize known outer membrane {beta}-barrel (OMBB) proteins and identify novel OMBBs in B. melitensis 16M. Proteins were modelled using five computational tools: AlphaFold 3, ESMFold, SWISS-MODEL, RoseTTAFold, and TrRosetta. Outer-membrane insertion of the novel OMBBs was confirmed using PPM 3.0, Protein GRAVY, DREAMM, and MemProtMD_Insane. Putative functions were predicted using structure- and sequence-based annotations. Sequence variation across 46 B. melitensis strains were identified and mapped onto the structural models. OMBB-associated protein complexes - the RND (Resistance-Nodulation-Division) efflux pumps, the lipopolysaccharide transport (Lpt) complex, and the {beta}-barrel assembly machinery (BAM) complex - were modelled, and protein-protein interactions (PPIs) were analyzed to confirm thermodynamically stable assemblies. This study presents a robust in silico strategy for exploring OMP architecture and provides valuable structural insights to support the development of diagnostics, targeted therapeutics, and vaccines against B. melitensis.