CEACAM mediated internalization of Opa proteoliposomes into human cells
Kuhn, Jason, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Columbus, Linda, As-Chemistry, University of Virginia
Carcinoembryonic antigen-like cell adhesion molecules (CEACAMs) are cell-surface proteins that are widely-expressed in the human body. In humans, CEACAM proteins play diverse roles in mediating cell adhesion, migration, and even host-pathogen interactions. Additionally, several CEACAM proteins exhibit increased expression on tumour cells in certain forms of pancreatic, gastric, hepatic, lung, and colorectal cancers. Because of the overexpression of CEACAM proteins on malignant cells, there is interest in developing strategies to target therapeutic delivery to cells overexpressing CEACAM proteins.
Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacteria that infect human host cells, including through binding to CEACAMs. Various surface proteins on Neisseria mediate bacterial engulfment into host cells. One such group of proteins is the opacity-associated (Opa) proteins on the outer membrane of Neisseria, which mediate Neisseria interactions with human CEACAM proteins. Previous work has shown that purified, recombinant Opa proteins can be folded into small unilamellar vesicles, termed liposomes, where the Opa proteins are functional. Because liposomes are common platforms for therapeutic delivery, this work investigates whether Opa-liposomes can be used to induce liposomal uptake into human cells through Opa-CEACAM interactions. Uptake of Opa-liposomes into cells through CEACAM would have relevance for the targeted delivery of therapeutic compounds into cancer cells overexpressing CEACAM proteins. This thesis details progress toward investigating Opa-liposome interactions with CEACAM.
Results indicate that cellular binding and uptake of Opa-liposomes can be assayed using imaging flow cytometry, with internalized liposomes able to be identified from surface-bound liposomes during data processing. Opa60 on liposomes promotes energy-dependent uptake of liposomes into HeLa cells when compared to control liposomes containing an Opa60 variant in which the binding region of Opa60 has been removed. Non-specific uptake of liposomes is described in the context of the HeLa cells used in these experiments, with particular emphasis on the relationship between liposome size and non-specific uptake. Results suggest that small liposomes can be non-specifically internalized into HeLa cells through bulk fluid-phase uptake during macropinocytosis.
Additionally, various considerations for the uptake of Opa-liposomes into CEACAM1+ HeLa cells are detailed. A positive correlation was found between progression through cell cycle and CEACAM1 expression, which results in increased Opa60-liposome internalization into HeLa cells later in the cell cycle that express higher amounts of CEACAM1. Following internalization, at least some Opa-liposomes are trafficked through the classical endocytic pathway, as shown through colocalization with markers for early endosomes and lysosomes. Finally, preliminary experiments are described in which Opa-liposomes are loaded with doxorubicin in order to investigate the Opa-mediated delivery of an active compound to cells.
PHD (Doctor of Philosophy)
CEACAM, Opa, liposome, targeted delivery, therapeutic delivery, cancer, imaging flow cytometry, Imagestream, proteoliposome
All rights reserved (no additional license for public reuse)