Development of a Ph-Triggered Cargo Delivery System with EsxA Mutants
Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis (Mtb). It was the leading cause of human death generated by a single infectious agent before the Coronavirus (COVID-19) pandemic. Mtb has a special mechanism to evade the host’s immune system and utilize macrophages as reservoirs. Latent TB infections have been a threat to public health. Liposome has been widely used for drug delivery. Immunoliposomes are special liposome carriers coated with ligands, such as antigens, targeting certain cells or organs to regulate the uptake of liposome. By constructing the liposome components, or surface modification, cargo release of liposome can be triggered with change of pH. EsxA is a small protein secreted by the Esx-1 secretion system with its chaperon EsxB. EsxA has a special membrane permeabilizing activity (MPA). Under low pH, EsxA/B goes through conformational change while EsxA exposes more hydrophobic residues, interrupting phagosome maturation and helping Mtb evade host immune response. Moreover, EsxA also interacts with the host’s immune system as a potent T-cell antigen and regulates host immune response through multiple pathways. In this study, we modified the liposome with EsxA mutants through a maleimide reaction. EsxA with G10C, G45C, and G88C mutations were generated and purified from both Escherichia coli (E. coli) and Mycobacterium smegmatis (M. smegmatis). During our experiment, the modified liposome system remained stable under physical pH. All EsxA mutants were able to rupture the liposome and trigger cargo release with drop of pH. G45C mutants showed best efficiency with even higher cargo release rate than free protein. EsxA/B heterodimers were not as efficient as EsxA alone. However, central linkage on G45C still enhanced the membrane rupture compared with free protein. This delivery system has potential for targeting delivery to bacteria reservoirs, and the enhancement of cargo release rate by central linkage may suggest a special role of central loop in membrane insertion.
Biochemistry|Biology|Cellular biology|Molecular biology
Chen, Shuchen, "Development of a Ph-Triggered Cargo Delivery System with EsxA Mutants" (2022). ETD Collection for University of Texas, El Paso. AAI29324481.