Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.
Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.
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DOI：
10.1016/j.xphs.2016.05.015
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发表时间：
2016-07
影响因子：
3.8
通讯作者：
Dickenson NE
中科院分区：
文献类型：
--
作者：
Bernard AR;Duarte SM;Kumar P;Dickenson NE
志贺氏菌依赖于III型分泌系统（T3 SS）作为入侵和定殖人类宿主的主要毒力因子。虽然估计有9000万志贺氏菌感染，每年造成全球超过10万人死亡，但分离和稳定许多T3 SS蛋白的挑战阻碍了对志贺氏菌入侵机制的全面了解，并进一步减缓了急需的志贺氏菌疫苗的进展。在这里，我们表明，非变性两性离子洗涤剂LDAO和非离子洗涤剂OPOE有效地分离的疏水志贺氏菌易位蛋白IpaC从共纯化的IpaC/IpgC伴侣结合的复合物。这两种洗涤剂导致单体IpaC表现出强的膜结合和裂解特性，而伴侣结合的复合物没有，这表明稳定洗涤剂提供了一种手段，以下IpaC的“激活”在体外。此外，生物物理表征发现，LDAO为IpaC提供了显着的热稳定性和时间稳定性，在室温下保护它几天，并短暂暴露于90°C的温度。总之，这项工作确定并表征了提供稳定的膜活性IpaC的条件，提供了对与膜的关键相互作用的深入了解，并为利用IpaC的天然免疫原性和LDAO提供的稳定性的未来疫苗制剂研究奠定了坚实的基础。
Shigella rely on a type III secretion system (T3SS) as the primary virulence factor for invasion and colonization of human hosts. While there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many T3SS proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent LDAO and non-ionic detergent OPOE efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC “activation” in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO.