Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Poster Abstracts

19-POS Board 5 INVESTIGATING THE ROLE OF LIPOPOLYSACCHARIDE IN OPACITY ASSOCIATED PROTEIN-HOST RECEPTOR SPECIFICITY AND STRUCTURE Connor M McDermott 1 ; Meagan Belcher Dufrisne 1 ; Linda Columbus 1 ; 1 University of Virginia, Chemistry, Charlottesville, VA, USA Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are a group of 12 cell surface receptors within the immunoglobulin family. Of the 12 members, four CEACAMs are pathogen binding: CEACAM1, CEACAM3, CEACAM5, and CEACAM6, and of those four, three CEACAMs: CEACAM1, CEACAM5, and CEACAM6, are well-known biomarkers for a variety of cancers. Almost all, CEACAM-ligand interactions occur at the conserved N-terminal domain including CEACAM-pathogen interactions. Neisseria gonorrhoeae (Ng) and Neisseria meningitidis (Nm) are the bacterial species which cause the sexual transmitted infection known as gonorrhea and bacterial meningitidis, respectively, and are both bacterial species that interact with human CEACAMs. These pathogenic Neisseria species are unique amongst CEACAM targeting pathogens because, unlike other pathogens that bind to CEACAM, Ng and Nm are able to exploit CEACAM binding to trigger uptake into non-phagocytic cells. This interaction is mediated by opacity-associated (Opa) proteins, which are eight-stranded outer membrane (OM) β -barrel proteins with four highly dynamic extracellular loops. Two of these loops contain regions of high amino acid sequence variability known as the hypervariable regions 1 (HV1) and 2 (HV2). Previous research has demonstrated that Opa-CEACAM specificity is dictated by the combination of specific HV sequences, however, the high sequence variability in the HV regions has prevented the identification of a CEACAM binding motif. Previous attempts to study specific Opa proteins in vitro have been unable to replicate the observed in vivo Opa-CEACAM specificity. We hypothesize the inability to recapitulate the in vivo specificity is because previous in vitro binding assays lacked a critical component of the Neisserial OM: lipooligosaccharide (LOS), which is a type of lipopolysaccharide (LPS) expressed by both pathogenic Neisseria species that lacks an O-antigen. Preliminary results show that Opa in the presence of LPS forms an SDS-resistant complex. Furthermore, using a microscale thermophoresis (MST) binding assay with Opa expressed to and subsequently extracted and purified from E. coli OM, we found that the monovalent binding affinity was weak (~ 2 µM). These results will not only help provide a greater understanding of the Neisseria pathogenesis, but provide an understanding of a mechanism that induces phagocytosis in non-phagocytic cells that may be of interest for receptor-mediated cellular uptake of foreign material such as therapeutics.

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