Omp33-36 loop mutations alter porin dynamics in resistant A. baumannii

Substitutions in the outer-membrane porin Omp33-36 altered the movement of channel-associated loops in two multidrug-resistant Acinetobacter baumannii isolates, according to a study combining clinical-isolate characterization, comparative structural modeling and atomistic membrane simulations. The protein’s overall 14-stranded beta-barrel remained stable, but the substitutions changed local packing, electrostatic interactions and hydrogen-bond networks near the channel entrance. Together with reduced expression of omp33-36, these changes could contribute to reduced antimicrobial permeability, although the study did not directly measure antibiotic flux or establish the mutations as an independent cause of resistance.

The researchers characterized two multidrug-resistant A. baumannii isolates, A_146 and A_106, recovered from the blood of septicemic neonates treated at a tertiary-care center in India. Multilocus sequence typing assigned the isolates to ST1406 and ST1488, respectively. Both organisms were resistant to multiple antimicrobial classes, including ceftazidime, cefepime, aztreonam, amikacin and ciprofloxacin, as determined by minimum inhibitory concentrations. The team sequenced omp33-36, quantified its expression by reverse-transcription quantitative PCR and screened the isolates for acquired resistance determinants, allowing the porin variants to be interpreted in the context of the organisms’ broader resistance phenotypes.

Relative to the susceptible reference strain ATCC 19606, omp33-36 expression was reduced to 0.3-fold in A_146 and 0.2-fold in A_106. Sequencing identified two distinct pairs of amino-acid substitutions in the porin. A_146 carried Phe125Asn and Glu243Ser, while A_106 carried Gly248Asp and Asp250Asn. All four positions occur in or near surface-exposed loop regions, where the protein’s ability to reorganize can influence the geometry and accessibility of the aqueous pathway through the outer membrane. Both isolates also carried blaPER and aac(6′)-Ib-cr, linking the analysis to a broader genetic background of resistance rather than an isolated porin defect.

To examine the structural consequences, the investigators built full-length models of the reference porin and the two variants. The experimentally determined Omp33-36 structure deposited as PDB 6GIE, which was solved by X-ray crystallography at 2.10-angstrom resolution, served as the principal template. Unresolved portions were modeled comparatively, and the clinical substitutions were introduced into the resulting structures. Ramachandran-plot analysis placed more than 90% of residues in favored or allowed regions, which the researchers considered evidence of acceptable stereochemical quality for the simulations. A broader comparison of 23 modeled porins from different Gram-negative organisms placed Omp33-36 within the conventional beta-barrel porin architecture while highlighting its unusually complex extracellular loops and extended periplasmic turns.

Omp33-36 differs from classical channels such as OmpF, in which a pore-forming loop helps create a constriction, and from substrate-selective porins such as OprD, which have distinctive loops around their channels. In Omp33-36, several loop and turn elements form a complex access environment around the transmembrane pathway. This arrangement means that a sequence substitution does not need to unfold or destroy the beta-barrel to affect channel behavior. A local change can instead alter steric separation, charge distribution, hydrogen bonding, the range of loop motions or the configurations available near the channel entrance. The clinical substitutions examined here were localized changes, so the investigators focused on whether they rewired interactions in these dynamic access regions while retaining the overall scaffold.

In A_146, Phe125Asn appears to act as a steric-to-polar switch. In the reference model, bulky Phe125 helps separate neighboring loop regions. Replacing it with the smaller, polar asparagine removes that steric constraint and can permit additional conformational rearrangement, including a proposed hydrogen bond between Asn125 and Asp250. The paired Glu243Ser substitution has a different physicochemical effect: it removes a negatively charged glutamate side chain from the Gly242-Gly252 loop and replaces it with a smaller hydroxyl-bearing serine. Ser243 can form short-range polar contacts with nearby residues such as Asn245 and Thr247. The combined model therefore suggests that Phe125Asn and Glu243Ser reshape the interaction network through complementary changes in steric freedom, charge and hydrogen bonding rather than through wholesale unfolding of the barrel.

A_106 produced a more extensive pattern of predicted cross-loop stabilization. Gly248Asp introduces a larger, negatively charged side chain at a position normally occupied by conformationally permissive glycine. The model predicts new hydrogen-bond and electrostatic contacts with residues in the neighboring Gly214-Glu216 region, including Gln215, Glu216 and Asn217. Asp250Asn replaces an exposed acidic residue with neutral asparagine, enabling additional polar contacts with regions containing Gln158-Asp162 and Gln182-Gln185. Together, these substitutions anchor the entrance-side loop to neighboring structural elements and restrict its range of motion. Conservation analysis showed that the altered positions are variable to intermediate in evolutionary conservation, and related substitutions occurred in some epidemic lineages, indicating that the study concerns particular substitution combinations in variable loop regions rather than resistance markers unique to ST1406 or ST1488.

The researchers embedded each modeled porin in a dipalmitoylphosphatidylcholine bilayer, added explicit water and counterions, and conducted all-atom molecular dynamics simulations. After energy minimization and staged constant-volume and constant-pressure equilibration, each system underwent a 100-nanosecond production run at 323 kelvin. Root mean square deviation showed that the wild-type and variant proteins reached stable overall conformations, while the mutants generally displayed smaller global fluctuations. Root mean square fluctuation revealed more complicated local behavior: some mutant loop residues oscillated frequently within geometrically constrained regions, while a reference loop could undergo a displacement that opened the lumen and then remain fixed. The radius of gyration stayed near 2.0 nanometers, with reported values of approximately 2.0 ± 0.5 nanometers, in all three systems, supporting preservation of the global fold despite transient local rearrangements.

These measurements show why global rigidity and local mobility should not be conflated. Root mean square deviation describes displacement from a reference conformation, root mean square fluctuation reflects residue-level movement, and radius of gyration tracks overall compactness. Their combined trajectories suggest that the substitutions did not destroy the beta-barrel. Instead, they redistributed conformational freedom: loop regions sampled different networks of contacts and sometimes moved closer to the channel pathway. The study interprets these changes as consistent with a working two-gate coupled-motion model. In that hypothesis, extracellular- and periplasm-facing access regions are spatially separated but dynamically coordinated components of one transmembrane route. Mutations may bias Omp33-36 toward conformations with less favorable access without creating a permanent physical barrier. The authors emphasize that “coupled motion” refers to coordinated behavior among multiple loop and turn elements, not to two individual gate-forming residues or proof that antibiotics open and close the channel in a synchronized sequence.

The findings remain a structural hypothesis rather than direct evidence of diminished antibiotic transport. No antibiotic flux was simulated or measured, pore geometry and hydration were not quantified with the resolution required to establish reduced permeability, and individual hydrogen bonds or lipid contacts were not evaluated for occupancy and energetic contribution. The elevated simulation temperature was intended to improve conformational sampling and did not reproduce the physiological temperature of bacterial growth. Reduced porin expression and sequence variation also occurred together in the clinical isolates, preventing separation of their effects, while the presence of other acquired resistance determinants means that Omp33-36 cannot be assigned responsibility for the multidrug-resistant phenotypes. Isogenic expression experiments, complementation, direct outer-membrane permeability assays, susceptibility measurements and molecular-flux calculations would be required to test the proposed mechanism. The study therefore identifies loop rewiring as a plausible contributor to permeability-related resistance in A. baumannii, not as an independently demonstrated mechanism or a basis for clinical recommendations.

Subject of Research: Omp33-36 loop substitutions and modeled changes in porin dynamics in multidrug-resistant Acinetobacter baumannii isolates

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

Article References: Roy, S., Ansari, K., Hazra, M., Junghare, V., Das, T., Samanta, A., Basu, S., & Hazra, S. (2026). Loop region mutations in Omp33-36: unveiling reduced antimicrobial permeability and resistance in Acinetobacter baumannii through structural modeling and membrane-protein simulations. Frontiers in Microbiology, 17, Article 1954565. https://doi.org/10.3389/fmicb.2026.1954565

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1954565

Keywords: Acinetobacter baumannii, antimicrobial resistance, Omp33-36, outer membrane, porin mutations, molecular dynamics, drug permeability, structural modeling, membrane protein, Loop, region, mutations