Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of .

EsxA and EsxB are secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of into the cytosol. Recent in vitro studies have suggested that the EsxAB heterodimer is dissociated upon acidification, which might allow EsxA insertion into lipid membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxAB heterodimer has not been detected through in vitro assays due to its negligible activity with DOPC liposomes. In this study, we established a new in vitro membrane assay to test the MPA activity of N-terminal acetylated EsxA (N-EsxA). We established that a dose-dependent increase in anionic charged lipids enhances the MPA of N-EsxA. The MPA of both N-EsxA and EsxAB were significantly increased with this new liposome system and made it possible to characterize the MPA of EsxAB in more physiologically-relevant conditions. We tested, for the first time, the effect of temperature on the MPA of N-EsxA and EsxAB in this new system. Interestingly, the MPA of N-EsxA was lower at 37 °C than at RT, and on the contrary, the MPA of EsxAB was higher at 37 °C than at RT. Surprisingly, after incubation at 37 °C, the MPA of N-EsxA continuously decreased over time, while MPA of EsxAB remained stable, suggesting EsxB plays a key role in stabilizing N-EsxA to preserve its MPA at 37 °C. In summary, this study established a new in vitro model system that characterizes the MPA of EsxAB and the role of EsxB at physiological-relevant conditions.