Characterization of EsxA:EsxB Heterodimer Membrane Interaction with a Novel POPC/POPG Liposome Model

Salvador Vazquez Vazquez Reyes, University of Texas at El Paso


EsxA and EsxB are co-secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of Mycobacterium tuberculosis into the cytosol. Current in vitro studies support a model in which the EsxA:EsxB heterodimer is dissociated upon acidification, which might allow EsxA insertion into liposome membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxA:EsxB heterodimer has not been detected with the DOPC liposome. In this study, we established a new in vitro membrane assay to test the MPA of EsxA and EsxB. We established that a dose-dependent increase in anionic charged lipids enhances the MPA of EsxA and EsxA:EsxB. The creation of a new liposome system made it possible to characterize the MPA of EsxA:EsxB at physiological temperature (37 °C). We have shown for the first time, the stabilizing role of EsxB in the MPA of EsxA at 37 °C. Encouraged by these findings, we tested the pH-dependent dissociation of the heterodimer. In previous studies, we have found evidence that EsxA is Nα-acetylated and that Post Translational Modifications (PTMs) play a role in the pH-dependent dissociation. However, we have not yet obtained direct evidence that the heterodimer dissociates upon acidification. Here, we designed two FRET assays to show the direct association of EsxA and EsxB at pH 7 and acidic pH. After which, we confirmed our results by comparing the association and kinetics of the non Nα-acetylated heterodimer (EsxA T2A: EsxB) with the EsxA:EsxB WT. Finally, we investigated the effects of EsxB C-terminus on EsxA:EsxB MPA. The C-terminus of EsxB C terminus plays a role in heterodimer co-secretion and attachment to monocytes. This suggests that the EsxB C terminus may be involved in the MPA of EsxA:EsxB. We have generated a C-terminal truncated EsxB mutant and used to test of the role of EsxB C terminus in EsxA mediated membrane disruption. In summary, this study established a new in vitro liposome model system that characterizes the MPA of EsxA:EsxB and the role of EsxB as a stabilizing protein at 37 °C.

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Recommended Citation

Vazquez Reyes, Salvador Vazquez, "Characterization of EsxA:EsxB Heterodimer Membrane Interaction with a Novel POPC/POPG Liposome Model" (2022). ETD Collection for University of Texas, El Paso. AAI29322534.