Quantitative Analysis of Neisserial Opa Protein Interactions with Human CEACAMS In Vitro 1168 views

Increasing bacterial resistance to antibiotics is an urgent global health concern. In order to combat this growing antibiotic resistance, alternative treatments must be developed. Developing new treatments requires a deeper understanding of bacterial pathogenesis. This dissertation presents investigations of the interaction between Opa proteins from Neisseria gonorrhoeae and Neisseria meningitidis, pathogens which cause gonorrhea and meningococcal meningitis respectively, and the human receptor CEACAM. Opa proteins are a family of 8-stranded β-barrel proteins found in the outer membrane of pathogenic Neisseria. Opa proteins contain regions of high sequence variation, which are involved in receptor interactions. The majority of Opa proteins interact with carcino-embryonic antigen-like cell adhesion molecules (CEACAMs), expressed on human host cell surfaces. Interactions of Opa proteins with CEACAM receptors are able to trigger engulfment of the bacterium into the host cell. While Opa proteins appear to selectively engage specific receptors, the molecular determinants of the interaction are unknown. Multiple sequence alignments of Opa protein sequences have not revealed a conserved receptor recognition motif. The investigations presented in this dissertation seek to gain a deeper understanding of Opa protein – CEACAM receptor interactions. Towards this end, an in vitro system was developed containing recombinant Opa proteins folded into liposomes. A selection of CEACAM binding Opa proteins were investigated for their ability to interact with the recombinant N-terminal domain of CEACAM receptors. Binding was qualitatively assessed using pull-down assays and immunoblotting to compare the specificity of Opa proteins in vitro to their in vivo function, and validate the Opa proteoliposome system for use in further experiments. Quantitative experiments were conducted to investigate the kinetics and thermodynamics of the Opa protein – CEACAM receptor interaction. A wider selection of Opa proteins were investigated for their affinity towards various CEACAM receptors. Tight affinities were calculated for all investigated interactions. Knowledge of Opa – CEACAM affinities is the first step towards gaining a deeper understanding of how Neisseria are able to trigger engulfment into host cells. Phagocytosis of the Opa proteoliposomes is being investigated to determine if there is a correlation between receptor specificity and bacterial engulfment. Various techniques are being utilized to gain insight into the mechanism of molecular recognition between Opa proteins and CEACAM receptors. Elucidating the molecular determinants of the Opa protein – CEACAM receptor interactions will provide the foundation for the design of a targeted liposome therapeutic delivery system for human cells.

PHD (Doctor of Philosophy)

English

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2016-04-28