VI型分泌系统(T6SS)是微生物-微生物和微生物-宿主相互作用的重要介质。革兰氏阴性菌使用T6SS注射T6SS效应物(T6Es),通常是具有毒性活性的蛋白质,进入邻近的细胞。抗菌效应物具有中和自我中毒的同源免疫蛋白。这里,我们应用新的结构生物信息学工具,从17,920个编码T6SS的细菌基因组数据集中,对T6Es及其同源免疫蛋白进行系统发现和功能注释.使用结构聚类,我们确定了517个推定的T6E家族,优于基于序列的聚类。我们开发了一个逻辑回归模型来可靠地量化新的T6E免疫对的蛋白质-蛋白质相互作用,产生517个T6E家族中231个的候选免疫蛋白。我们使用了敏感的基于结构的注释,为51%的T6E家族提供了功能注释,再次优于基于序列的注释。接下来,我们在大肠杆菌中使用基础实验验证了四种新型T6E免疫对。特别是,我们表明Pfam结构域DUF3289是ColicinM的同源物,DUF943充当其同源免疫蛋白。此外,我们发现了一种新的T6E,它是SleB的结构同源物,裂解性转糖基酶,并确定了一种特定的谷氨酸盐作为其推定的催化残基。总的来说,这项研究将新的结构生物信息学工具应用于T6E-免疫对的发现,并提供了带注释的T6E免疫对的广泛数据库。
The type VI secretion system (T6SS) is an important mediator of microbe-microbe and microbe-host interactions. Gram-negative bacteria use the T6SS to inject T6SS effectors (T6Es), which are usually proteins with toxic activity, into neighboring cells. Antibacterial effectors have cognate immunity proteins that neutralize self-intoxication. Here, we applied novel structural bioinformatic tools to perform systematic discovery and functional annotation of T6Es and their cognate immunity proteins from a dataset of 17,920 T6SS-encoding bacterial genomes. Using structural clustering, we identified 517 putative T6E families, outperforming sequence-based clustering. We developed a logistic regression model to reliably quantify protein-protein interaction of new T6E-immunity pairs, yielding candidate immunity proteins for 231 out of the 517 T6E families. We used sensitive structure-based annotation which yielded functional annotations for 51% of the T6E families, again outperforming sequence-based annotation. Next, we validated four novel T6E-immunity pairs using basic experiments in E. coli. In particular, we showed that the Pfam domain DUF3289 is a homolog of Colicin M and that DUF943 acts as its cognate immunity protein. Furthermore, we discovered a novel T6E that is a structural homolog of SleB, a lytic transglycosylase, and identified a specific glutamate that acts as its putative catalytic residue. Overall, this study applies novel structural bioinformatic tools to T6E-immunity pair discovery, and provides an extensive database of annotated T6E-immunity pairs.