Centre for Molecular Microbiology and Infection (CMMI), Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK.
Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.
Microbiology (Reading). 2011 Dec;157(Pt 12):3292-3305. doi: 10.1099/mic.0.051987-0. Epub 2011 Aug 26.
Bacterial pathogens use a range of protein secretion systems to colonize their host. One recent addition to this arsenal is the type VI secretion system (T6SS), which is found in many Gram-negative bacteria. The T6SS involves 12-15 components, including a ClpV-like AAA(+) ATPase. Moreover, the VgrG and Hcp components have been proposed to form a puncturing device, based on structural similarity to the tail spike components gp5/gp27 and the tail tube component gp19 of the T4 bacteriophage, respectively. Another T6SS component shows similarity to a T4 phage protein, namely gp25. The gp25 protein has been proposed to have lysozyme activity. Other T6SS components do not exhibit obvious similarity to characterized T4 phage components. The genome of Pseudomonas aeruginosa contains three T6SS gene clusters. In each cluster a gene encoding a putative member of the gp25-like protein family was identified, which we called HsiF. We confirmed this similarity by analysing the structure of the P. aeruginosa HsiF proteins using secondary and tertiary structure prediction tools. We demonstrated that HsiF1 is crucial for the T6SS-dependent secretion of Hcp and VgrG. Importantly, lysozyme activity of HsiF proteins was not detectable, and we related this observation to the demonstration that HsiF1 localizes to the cytoplasm of P. aeruginosa. Finally, our data showed that a conserved glutamate, predicted to be required for proper HsiF folding, is essential for its function. In conclusion, our data confirm the central role of HsiF in the T6SS mechanism, provide information on the predicted HsiF structure, and call for reconsideration of the function of gp25-like proteins.
细菌病原体利用一系列蛋白质分泌系统来定殖其宿主。最近在这个武器库中增加了一种类型的 VI 型分泌系统（T6SS），它存在于许多革兰氏阴性菌中。T6SS 涉及 12-15 个组件，包括 ClpV 样 AAA(+)ATP 酶。此外，VgrG 和 Hcp 组件被认为基于与 T4 噬菌体的尾刺组件 gp5/gp27 和尾管组件 gp19 的结构相似性，分别形成一个穿孔装置。另一个 T6SS 组件与 T4 噬菌体蛋白具有相似性，即 gp25。gp25 蛋白被认为具有溶菌酶活性。其他 T6SS 组件与特征化的 T4 噬菌体组件没有明显的相似性。铜绿假单胞菌的基因组包含三个 T6SS 基因簇。在每个簇中，都鉴定出一个编码 gp25 样蛋白家族的推定成员的基因，我们称之为 HsiF。我们通过使用二级和三级结构预测工具分析铜绿假单胞菌 HsiF 蛋白的结构来证实这种相似性。我们证明 HsiF1 对于 T6SS 依赖的 Hcp 和 VgrG 分泌是至关重要的。重要的是，HsiF 蛋白的溶菌酶活性无法检测到，我们将这一观察结果与 HsiF1 定位于铜绿假单胞菌细胞质的证明联系起来。最后，我们的数据表明，一个保守的谷氨酸，预测需要适当的 HsiF 折叠，对其功能至关重要。总之，我们的数据证实了 HsiF 在 T6SS 机制中的核心作用，提供了有关预测 HsiF 结构的信息，并呼吁重新考虑 gp25 样蛋白的功能。
