Loop region mutations in Omp33-36: unveiling reduced antimicrobial permeability and resistance in Acinetobacter baumannii through structural modeling and membrane-protein simulations

Background Acinetobacter baumannii can reduce its susceptibility to antimicrobials by altering the permeability of the outer membrane, e.g., by changing the abundance and function of porins. Omp33-36 has been implicated in susceptibility to multiple antibiotics including carbapenems, but the structural effect of sequence variations observed in clinical isolates is poorly understood. We investigated whether substitutions in Omp33-36 from multidrug-resistant clinical isolates affect porin dynamics and contribute to permeability-related resistance. Methods Multilocus sequence typing identified lineages and Sanger sequencing identified Omp33-36 substitutions in A. baumannii bloodstream isolates. The expression of omp33-36 was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and the acquired resistance determinants were analyzed by PCR. Based on the experimentally determined structure (PDB 6GIE), we built full-length wild-type and variant models of Omp33-36, embedded them into a phospholipid bilayer and performed all-atom molecular dynamics simulations to study their conformational behavior, residue mobility and loop-associated interactions. Results Two multidrug-resistant isolates with reduced omp33-36 expression harbored substitutions Phe125Asn/Glu243Ser (A_146) and Gly248Asp/Asp250Asn (A_106). The β-barrel scaffold was stable, but substitutions affected local packing, interaction network and loop dynamics. Phe125Asn reduced steric constraints and allowed more polar interactions. Mutations in the Gly242-Gly252 region created new local and cross-region contacts that constrained conformational freedom. These changes favored conformational states with increased mechanical constraints and without global destabilization. The observations are consistent with a working two-gate coupled motion model in which extracellular- and periplasmic-facing access regions are dynamically coupled components. The mutation-associated change in loop dynamics and preference for less-accessible channel states may disrupt coordination, but no direct evidence for diminished antibiotic flux was provided. Conclusion Substitutions in Omp33-36 alter local networks of interactions and loop dynamics but preserve the β-barrel architecture. These changes and a reduced expression of porins may result in changes in permeability. However, the presence of other resistance determinants makes Omp33-36 changes a possible contributor to a multifactorial resistance phenotype rather than an independently demonstrated cause. Direct measurements of permeability and isogenic studies with expression control are necessary to validate the proposed mechanism.