The type VI secretion system (T6SS) is a contractile injection apparatus that translocates a spike loaded with various effectors directly into eukaryotic and prokaryotic target cells. Such T6SS spike consists of a needle-shaped trimer of VgrG proteins topped by a conical and sharp PAAR protein that facilitates puncturing of the target membrane. T6SS-delivered effector proteins can be either fused to one of the two spike proteins or interact with either in a highly specific manner. In Agrobacterium tumefaciens the T6SS effector Tde1 is targeted to its cognate VgrG1 protein. Here, we attempted to use a VgrG shuttle to deliver a heterologous T6SS effector by directing Tde1 onto a T6SS spike in Pseudomonas aeruginosa. For this, we designed chimeras between VgrG1 from A. tumefaciens and VgrG1a from P. aeruginosa and showed that modification of the spike protein hampered T6SS functionality in the presence of the Tde1 effector complex. We provide evidence suggesting that Tde1 specifically binds to the VgrG spike in the heterologous environment and propose that there are additional requirements to allow proper effector delivery and translocation. Our work sheds light on complex aspects of the molecular mechanisms of T6SS delivery and highlights some limitations on how effectors can be translocated using this nanomachine.

VI 型分泌系统 (T6SS) 是一种收缩注射装置，可将装载各种效应器的尖峰直接转移到真核和原核靶细胞中。这种 T6SS 尖峰由 VgrG 蛋白的针状三聚体组成，顶部是锥形和尖锐的 PAAR 蛋白，有助于刺穿靶膜。T6SS 传递的效应蛋白可以与两种刺突蛋白中的一种融合，也可以以高度特异性的方式与其中一种相互作用。在根癌农杆菌T6SS 效应器 Tde1 靶向其同源 VgrG1 蛋白。在这里，我们尝试使用 VgrG 穿梭机通过将 Tde1 引导到 T6SS 尖峰上来传递异源 T6SS 效应器。铜绿假单胞菌. 为此，我们设计了 VgrG1 之间的嵌合体根癌农杆菌和 VgrG1a 来自铜绿假单胞菌并表明在 Tde1 效应复合物存在的情况下，刺突蛋白的修饰阻碍了 T6SS 的功能。我们提供的证据表明 Tde1 在异源环境中与 VgrG 尖峰特异性结合，并提出有额外的要求来允许适当的效应器传递和易位。我们的工作揭示了 T6SS 传递的分子机制的复杂方面，并强调了使用这种纳米机器如何转移效应器的一些限制。