Unlocking Bacterial Enigmas: The LA27 Dynamic Journey Through LOS/LPS-Embedded Membrane Barriers

Gram-negative bacteria, which include clinically and economically significant pathogens such as Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, possess a unique outer membrane that plays a crucial role in pathogenicity and antibiotic resistance. This membrane is characterized by an asymmetric bilayer architecture where the inner leaflet consists primarily of phospholipids and the outer leaflet is enriched with lipopolysaccharides (LPS) or lipooligosaccharides (LOS). The complex molecular structure of LPS, comprising lipid A, the core oligosaccharide, and the O-antigen, makes it a critical determinant of bacterial virulence and an ideal target for selective biosensing applications. In light of the limitations of conventional LPS detection assays such as the Limulus Amebocyte Lysate (LAL) test, which present cost and ethical concerns, aptamer-based detection systems have emerged as a promising alternative. Aptamers are synthetic oligonucleotides that exhibit high binding affinity and specificity toward their targets. In this study, we investigated the molecular interaction of the LA27 aptamer, previously shown to bind LPS with a dissociation constant (Kd) of approximately 46.2 ± 9.5 nM, with biomimetic membranes that model the bacterial outer envelope. Three different bacterial LOS/LPS molecules, extracted from Akkermansia, Flavobacterium, and Paenalcaligenes Hominis, were used to construct asymmetric supported lipid bilayers (SLBs) and symmetric large unilamellar vesicles (LUVs). The resulting biomimetic systems were characterized using neutron reflectometry (NR), small-angle neutron scattering (SANS), and dynamic light scattering (DLS) to probe the structural perturbations induced upon interaction with the LA27 aptamer. NR experiments revealed that the intrinsic asymmetry of the bilayers was maintained, with distinct layer thicknesses and hydration levels observed depending on the LOS/LPS composition. In particular, the Akkermansia LOS-containing bilayers exhibited high hydration in the phosphocholine regions and significant perturbations after exposure to LA27, suggesting an ability of the aptamer to penetrate the bilayer. Conversely, Flavobacterium LOS and Paenalcaligenes Hominis LPS systems displayed lower degrees of perturbation; the latter, in particular, showed evidence of the O-antigen moiety hindering deep aptamer penetration and favoring surface adhesion. SANS and DLS studies on LUV models further supported these findings. For Akkermansia LOS-containing vesicles, both the lamellar thickness and hydrodynamic radius remained largely unaltered after aptamer exposure, consistent with penetration and a resulting minimal impact on the vesicle’s external dimensions. In contrast, vesicles incorporating Flavobacterium LOS and Paenalcaligenes Hominis LPS demonstrated notable increases in both lamellar thickness and hydrodynamic radius. These alterations are interpreted as a consequence of LA27 adhesion predominantly occurring at the vesicle surface, thereby inducing morphological changes. Collectively, these results elucidate the molecular mechanisms underlying the biorecognition of LOS/LPS by the LA27 aptamer. The differential interaction modes, ranging from membrane penetration to surface adhesion, are modulated by the chemical and structural characteristics of the LOS/LPS molecules. The integrated use of neutron scattering and light scattering techniques provides a robust framework for the investigation of nano-bio-interface phenomena and underscores the potential of aptamer-based platforms in the selective biosensing of bacterial endotoxins.