Department of Biochemistry, University of Oxfordgrid.4991.5, Oxford, United Kingdom.
Department of Zoology, University of Oxfordgrid.4991.5, Oxford, United Kingdom.
mBio. 2022 Apr 26;13(2):e0339621. doi: 10.1128/mbio.03396-21. Epub 2022 Mar 28.
Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent among these are contact-dependent inhibition (CDI), type VI secretion (T6SS), and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain, that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa-specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact-dependent and contact-independent means. Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain that is essential for inner membrane translocation and is widespread in other bacterial competition systems. Additionally, the presented data link two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics.
细菌利用多种攻击策略在生态位中获得优势。其中突出的有接触依赖抑制（CDI）、VI 型分泌系统（T6SS）和细菌素。这些系统的细胞毒性终点通常是将核酸内切酶递送到细胞质中。革兰氏阴性细菌细胞质膜的这种核酸内切酶如何易位尚不清楚。在这里，我们鉴定了一个小的、保守的 15kDa 结构域，我们称之为内膜易位（IMT）结构域，它普遍存在于 T6SS 和细菌素中，并与核酸内切酶效应结构域相连。通过使用完整细胞和球形体细胞的荧光显微镜检测，我们证明了铜绿假单胞菌特异性细菌素 pyocin G（PyoG）的 IMT 结构域对于毒素核酸内切酶结构域向细胞质的导入是必需的。我们还表明，PyoG 易位到细胞质依赖于内膜蛋白 FtsH（一种 AAA+ATPase/蛋白酶）和 TonB1，后者通常与细菌素穿过外膜的运输有关。我们的研究揭示了 IMT 结构域指导 PyoG 的细胞毒性核酸内切酶穿过细胞质膜，更广泛地说，已经适应了通过接触依赖和非接触依赖的方式将其他毒性核酸内切酶递送到革兰氏阴性细菌的运输。核酸内切细菌素是治疗抗生素耐药细菌感染的潜在抗菌药物。虽然外膜易位的机制开始被理解，但内膜转运的机制尚不清楚。本研究使用 PyoG 作为模型核酸内切细菌素，并定义了一个保守结构域，该结构域对于内膜易位是必需的，并且在其他细菌竞争系统中广泛存在。此外，所提供的数据将两个膜蛋白 FtsH 和 TonB1 与 PyoG 的内膜易位联系起来。这些发现表明，该结构域对于通过不同和不同细菌竞争系统传递的核酸内切酶的细胞摄取机制具有普遍重要性。这项工作对于新蛋白抗生素的设计也很重要。
