Division of Infectious Diseases, Department of Medicine (Microbiology and Molecular Genetics), Massachusetts General Hospital and Harvard Medical School, Cambridge, Massachusetts, USA.
mBio. 2012 Feb 14;3(1). doi: 10.1128/mBio.00243-11. Print 2012.
Many Gram-negative bacteria utilize specialized secretion systems to inject proteins (effectors) directly into host cells. Little is known regarding how bacteria ensure that only small subsets of the thousands of proteins they encode are recognized as substrates of the secretion systems, limiting their identification through bioinformatic analyses. Many of these proteins require chaperones to direct their secretion. Here, using the newly described protein interaction platform assay, we demonstrate that type 3 secretion system class IB chaperones from one bacterium directly bind their own effectors as well as those from other species. In addition, we observe that expression of class IB homologs from seven species, including pathogens and endosymbionts, mediate the translocation of effectors from Shigella directly into host cells, demonstrating that class IB chaperones are often functionally interchangeable. Notably, class IB chaperones bind numerous effectors. However, as previously proposed, they are not promiscuous; rather they recognize a defined sequence that we designate the conserved chaperone-binding domain (CCBD) sequence [(LMIF)(1)XXX(IV)(5)XX(IV)(8)X(N)(10)]. This sequence is the first defined amino acid sequence to be identified for any interspecies bacterial secretion system, i.e., a system that delivers proteins directly into eukaryotic cells. This sequence provides a new means to identify substrates of type III secretion systems. Indeed, using a pattern search algorithm for the CCBD sequence, we have identified the first two probable effectors from an endosymbiont, Sodalis glossinidius.
Many Gram-negative pathogens utilize type 3 secretion systems to deliver tens of effectors into host cells. In order to understand the diverse ways that these organisms cause disease, it is necessary to identify their effectors, many of which require chaperones to be secreted. Here we establish that class IB chaperones are not promiscuous, as previously proposed, but rather recognize a conserved effector sequence. We demonstrate that pattern search algorithms based on this defined sequence can be used to identify previously unknown effectors. Furthermore, we observe that class IB chaperones from at least seven bacterial species are functionally interchangeable. Not only do they bind and mediate the delivery of their own set of effectors into host cells but they also bind to type 3 substrates from other bacteria, suggesting that inhibitors that block chaperone-effector interactions could provide a novel means to effectively treat infections due to Gram-negative pathogens, including organisms resistant to currently available antibiotics.
许多革兰氏阴性细菌利用专门的分泌系统将蛋白质（效应物）直接注入宿主细胞。关于细菌如何确保它们编码的数千种蛋白质中只有一小部分被识别为分泌系统的底物，从而限制了通过生物信息学分析对其进行鉴定，目前我们知之甚少。许多这类蛋白质需要伴侣来指导它们的分泌。在这里，我们使用新描述的蛋白质相互作用平台测定法，证明一种细菌的 III 型分泌系统类 IB 伴侣蛋白直接与自身效应物以及其他物种的效应物结合。此外，我们观察到来自七个物种（包括病原体和内共生体）的类 IB 同源物的表达介导了痢疾志贺菌的效应物直接转位进入宿主细胞，表明类 IB 伴侣蛋白通常在功能上是可互换的。值得注意的是，类 IB 伴侣蛋白结合了许多效应物。然而，正如之前提出的，它们并非杂乱无章；相反，它们识别一个已定义的序列，我们将其命名为保守伴侣结合域（CCBD）序列 [(LMIF)(1)XXX(IV)(5)XX(IV)(8)X(N)(10)]。这是首次为任何种间细菌分泌系统（即直接将蛋白质递送至真核细胞的系统）定义的氨基酸序列。该序列为鉴定 III 型分泌系统的底物提供了新的手段。事实上，我们使用 CCBD 序列的模式搜索算法，鉴定了内共生体 Sodalis glossinidius 的前两个可能的效应物。
许多革兰氏阴性病原体利用 III 型分泌系统将数十种效应物递送至宿主细胞。为了了解这些生物体引起疾病的多种方式，有必要鉴定它们的效应物，其中许多需要伴侣蛋白来分泌。在这里，我们确定类 IB 伴侣蛋白并非如之前提出的那样杂乱无章，而是识别一个保守的效应物序列。我们证明，基于此定义序列的模式搜索算法可用于鉴定以前未知的效应物。此外，我们观察到至少七种细菌物种的类 IB 伴侣蛋白在功能上是可互换的。它们不仅结合并介导自身效应物进入宿主细胞，还结合来自其他细菌的 III 型底物，这表明抑制伴侣-效应物相互作用的抑制剂可能为有效治疗革兰氏阴性病原体引起的感染提供一种新方法，包括对现有抗生素具有耐药性的生物体。
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