Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from pefE, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.

VI型分泌系统（T6SS）的功能是将致命的有效载荷输送到目标细胞中。许多研究表明，针对单个致死性T6SS效应蛋白的保护需要关联的解毒剂免疫蛋白，这两种蛋白通常在两个基因的操纵子中一起编码。T6SS和效应子-免疫对足以杀死和免疫。在本文中，我们描述了一个T6SS效应子操纵子，它不同于传统的效应子-免疫对，因为致命的效应子功能需要八个基因，但可以被一个免疫蛋白所抵消。在这项研究中，我们调查了PefE T6SS免疫蛋白在两个具有几乎相同的效应子操纵子的菌株之间的识别中所起的作用。有趣的是，尽管包含八种相同的效应蛋白中的七种，保守程度较低的免疫蛋白仅提供针对其天然效应子的保护，这表明特异性和识别性可能取决于免疫蛋白和一种效应子基因产物内的变异。可变效应子基因产物PefD在上游编码pefE，并显示可被PefE抵消的毒性活性，而与T6SS活性无关。有趣的是，在整个PEF操纵子是必要的经由T6SS发挥毒性活性P。奇异的，单独产生PefD和PefE不能发挥这种效应子活性。由两个P构建的嵌合PefE蛋白。奇迹菌株用于将免疫功能定位于三个氨基酸。使用定点诱变将这些残基从一种变体改变为另一种，从而产生了混杂免疫蛋白。这些发现支持了这样一种观念，即保守效应子之间的细微差异足以满足T6SS介导的亲属歧视，并且PefD需要其他因素才能发挥T6SS依赖性效应子的作用。