State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Department of Ecosystem and Public Health, University of Calgary, Calgary, Alberta, Canada.
Appl Environ Microbiol. 2021 May 26;87(12):e0043521. doi: 10.1128/AEM.00435-21.
The type VI secretion system (T6SS) is a widespread weapon employed by Gram-negative bacteria for interspecies interaction in complex communities. Analogous to a contractile phage tail, the double-tubular T6SS injects toxic effectors into prokaryotic and eukaryotic neighboring cells. Although effectors dictate T6SS functions, their identities remain elusive in many pathogens. Here, we report the lysozyme-like effector TseP in Aeromonas dhakensis, a waterborne pathogen that can cause severe gastroenteritis and systemic infection. Using secretion, competition, and enzymatic assays, we demonstrate that TseP is a T6SS-dependent effector with cell wall-lysing activities, and TsiP is its cognate immunity protein. Triple deletion of and two known effector genes, and , abolished T6SS-mediated secretion, while complementation with any single effector gene partially restored bacterial killing and Hcp secretion. In contrast to whole-gene deletions, the triple-effector inactivation in the 3eff mutant abolished antibacterial killing but not T6SS secretion. We further demonstrate that the 3eff mutation abolished T6SS-mediated toxicity of SSU to Dictyostelium discoideum amoebae, suggesting that the T6SS physical puncture is nontoxic to eukaryotic cells. These data highlight not only the necessity of possessing functionally diverse effectors for survival in multispecies communities but also that effector inactivation would be an efficient strategy to detoxify the T6SS while preserving its delivery efficiency, converting the T6SS to a platform for protein delivery to a variety of recipient cells. Delivery of cargo proteins via protein secretion systems has been shown to be a promising tool in various applications. However, secretion systems are often used by pathogens to cause disease. Thus, strategies are needed to detoxify secretion systems while preserving their efficiency. The T6SS can translocate proteins through physical puncture of target cells without specific surface receptors and can target a broad range of recipients. In this study, we identified a cell wall-lysing effector, and by inactivating it and the other two known effectors, we have built a detoxified T6SS-active strain that may be used for protein delivery to prokaryotic and eukaryotic recipient cells.
VI 型分泌系统（T6SS）是革兰氏阴性细菌在复杂群落中进行种间相互作用的广泛武器。类似于收缩性噬菌体尾部，双管状 T6SS 将毒性效应器注入原核和真核邻近细胞。尽管效应物决定了 T6SS 的功能，但在许多病原体中，它们的身份仍然难以捉摸。在这里，我们报告了水生病原体 Aeromonas dhakensis 中的溶菌酶样效应物 TseP。它可以引起严重的肠胃炎和全身感染。通过分泌、竞争和酶测定，我们证明 TseP 是一种依赖于 T6SS 的效应物，具有细胞壁裂解活性，而 TsiP 是其同源免疫蛋白。和两个已知效应基因和的三缺失消除了 T6SS 介导的分泌，而用任何单个效应基因进行互补部分恢复了细菌杀伤和 Hcp 分泌。与全基因缺失相比，3eff 突变体中的三重效应物失活消除了抗菌杀伤但没有消除 T6SS 分泌。我们进一步证明，3eff 突变体消除了 T6SS 介导的 SSU 对 Dictyostelium discoideum 变形虫的毒性，表明 T6SS 的物理穿孔对真核细胞没有毒性。这些数据不仅强调了在多物种群落中生存需要具有功能多样化的效应物，而且还表明效应物失活将是一种有效的解毒 T6SS 的策略，同时保持其输送效率，将 T6SS 转化为将货物蛋白递送至各种受体细胞的平台。通过蛋白质分泌系统输送货物蛋白已被证明是各种应用中的一种有前途的工具。然而，分泌系统经常被病原体用来引起疾病。因此，需要策略来解毒分泌系统，同时保持其效率。T6SS 可以通过物理穿孔靶细胞而无需特定的表面受体来转运蛋白质，并可以靶向广泛的受体。在这项研究中，我们鉴定了一种细胞壁裂解效应物，通过使该效应物及其它两种已知效应物失活，我们构建了一种解毒的 T6SS 活性菌株，可用于将蛋白质递送至原核和真核受体细胞。
