Vazquez Reyes Salvador, Ray Supriyo, Aguilera Javier, Sun Jianjun
Department of Biological Sciences, University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968, USA.
Border Biomedical Research Center at University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968, USA.
Pathogens. 2020 Dec 2;9(12):1015. doi: 10.3390/pathogens9121015.
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.
EsxA和EsxB以异二聚体形式分泌，并且已证明它们在吞噬体破裂和进入细胞质溶胶的转运过程中发挥关键作用。最近的体外研究表明，EsxAB异二聚体在酸化时会解离，这可能使EsxA插入脂质膜中。虽然EsxA的膜通透活性（MPA）在由二油酰磷脂酰胆碱（DOPC）组成的脂质体中已得到充分表征，但由于EsxAB异二聚体与DOPC脂质体的活性可忽略不计，尚未通过体外试验检测到其MPA。在本研究中，我们建立了一种新的体外膜试验来测试N端乙酰化的EsxA（N-EsxA）的MPA活性。我们发现带负电荷的脂质的剂量依赖性增加会增强N-EsxA的MPA。使用这种新的脂质体系统，N-EsxA和EsxAB的MPA均显著增加，这使得在更生理相关的条件下表征EsxAB的MPA成为可能。我们在这个新系统中首次测试了温度对N-EsxA和EsxAB的MPA的影响。有趣的是，N-EsxA在37℃时的MPA低于室温，相反，EsxAB在37℃时的MPA高于室温。令人惊讶的是，在37℃孵育后，N-EsxA的MPA随时间持续下降，而EsxAB的MPA保持稳定，这表明EsxB在稳定N-EsxA以在37℃保持其MPA方面起关键作用。总之，本研究建立了一个新的体外模型系统，该系统表征了EsxAB的MPA以及EsxB在生理相关条件下的作用。
