The Breadth and Molecular Basis of Hcp-Driven Type VI Secretion System Effector Delivery.
The Breadth and Molecular Basis of Hcp-Driven Type VI Secretion System Effector Delivery.
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DOI：
10.1128/mbio.00262-21
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发表时间：
2021-06-29
期刊：
影响因子：
6.4
通讯作者：
Filloux A
中科院分区：
文献类型：
--
作者：
Howard SA;Furniss RCD;Bonini D;Amin H;Paracuellos P;Zlotkin D;Costa TRD;Levy A;Mavridou DAI;Filloux A
VI型分泌系统(T6SS)是一种细菌纳米级武器，可以将毒素输送到从细菌和真菌到动物宿主的各种猎物中。该系统的胞浆收缩鞘包裹着堆积的六聚体Hcp蛋白环，形成内管。在这个试管的顶端是一个穿刺器，该穿刺器包括一个三聚体VgrG，顶端是一个单体PAR蛋白。由于效应器可以加载到T6SS设备的不同位置，特别是内管和穿刺器，因此单个系统在每个发射事件中释放的毒素数量仍然未知。每个VgrG或PAAR可以绑定一个效应器，并且可以在Hcp环腔中携带额外的效应器货物。虽然许多VgrG和PAAR结合的毒素已经被表征，但到目前为止，已知的Hcp结合效应因子很少。在这里，我们使用了3个已知的铜绿假单胞菌Hcp蛋白(Hcp1至-3)，每个蛋白都与该生物中的三个T6SS之一(H1-T6SS、H2-T6SS和H3-T6SS)相关联，进行了体内下拉分析。我们证实了Hcp1与TSE1 to-4的已知相互作用，进一步合成了Hcp1-Tse4复合体，并确定了潜在的Hcp1结合的新效应子。此外，我们还证明了Hcp2和Hcp3可以穿梭T6SS对大肠杆菌有毒性的货物。最后，我们使用TSE1-Bla嵌合体来探索Hcp乘客的装载策略，发现虽然大的效应器可以加载到Hcp上，但形成的复合体堵塞了系统，取消了T6SS的功能。
The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2-T6SS, and H3-T6SS), to perform in vivo pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coli. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function.