Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context.
Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context.
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DOI：
10.7554/elife.79796
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发表时间：
2022-06-28
期刊：
影响因子：
7.7
通讯作者：
Chou, Seemay
中科院分区：
文献类型：
--
作者：
Radkov, Atanas;Sapiro, Anne L.;Flores, Sebastian;Henderson, Corey;Saunders, Hayden;Kim, Rachel;Massa, Steven;Thompson, Samuel;Mateusiak, Chase;Biboy, Jacob;Zhao, Ziyi;Starita, Lea M.;Hatleberg, William L.;Vollmer, Waldemar;Russell, Alistair B.;Simorre, Jean-Pierre;Anthony-Cahill, Spencer;Brzovic, Peter;Hayes, Beth;Chou, Seemay
毒素的细菌T6SS酰胺酶效应子（Tae）超家族的成员在竞争细菌之间递送以降解细胞壁肽聚糖。虽然Taes具有共同的底物，但它们在不同的竞争物种中表现出不同的抗菌效力。为了研究这些差异的分子基础，我们使用核磁共振和称为深度突变扫描（DMS）的高通量体内遗传方法相结合，定量定义了铜绿假单胞菌PAO1的Tae1的功能决定簇。正如预期的那样，综合分析证实了Tae1催化中心附近的关键残基的作用。出乎意料的是，DMS揭示了从一个更大的，环状的功能热点周围延伸的酶的整个圆周的酶活性的实质性贡献。不同生长条件下的比较DMS强调了不同表面的功能贡献如何高度依赖于环境，随着靶细胞壁的组成而变化。这些观察结果表明，Tae1与完整的细胞壁网络，通过一个更分散的三维相互作用界面比以前认识到，提供了一个解释不同的革兰氏阴性竞争者之间的抗菌效力观察到的差异。几个Tae1变体与其同源免疫蛋白的进一步结合研究表明，保持保护Tae活性的要求可能是野生Tae1毒性突变景观的重要限制。总的来说，我们的工作表明，Tae多样化可能是由多个独立的压力形成的，以保持与不同细菌物种和条件的结合伴侣的相互作用。
Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.