Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts, USA.
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
mBio. 2018 Jun 19;9(3):e01050-18. doi: 10.1128/mBio.01050-18.
Numerous Gram-negative bacterial pathogens utilize type III secretion systems (T3SSs) to inject tens of effector proteins directly into the cytosol of host cells. Through interactions with cognate chaperones, type III effectors are defined and recruited to the sorting platform, a cytoplasmic component of these membrane-embedded nanomachines. However, notably, a comprehensive review of the literature reveals that the secretion of most type III effectors has not yet been linked to a chaperone, raising questions regarding the existence of unknown chaperones as well as the universality of chaperones in effector secretion. Here, we describe the development of the first high-throughput type III secretion (T3S) assay, a semiautomated solid-plate-based assay, which enables the side-by-side comparison of secretion of over 20 effectors under a multitude of conditions. Strikingly, we found that the majority of effectors are secreted at equivalent levels by wild-type and variants of that no longer encode one or all known T3S effector chaperones. In addition, we found that effectors are efficiently secreted from a laboratory strain of expressing the core type III secretion apparatus (T3SA) but no other specific proteins. Furthermore, we observed that the sequences necessary and sufficient to define chaperone-dependent and -independent effectors are fundamentally different. Together, these findings support the existence of a major, previously unrecognized, noncanonical chaperone-independent secretion pathway that is likely common to many T3SSs. Many bacterial pathogens use specialized nanomachines, including type III secretion systems, to directly inject virulence proteins (effectors) into host cells. Here, we present the first extensive analysis of chaperone dependence in the process of type III effector secretion, providing strong evidence for the existence of a previously unrecognized chaperone-independent pathway. This noncanonical pathway is likely common to many bacteria, as an extensive review of the literature reveals that the secretion of multiple type III effectors has not yet been knowingly linked to a chaperone. While additional studies will be required to discern the molecular details of this pathway, its prevalence suggests that it can likely serve as a new target for the development of antimicrobial agents.
许多革兰氏阴性细菌病原体利用 III 型分泌系统（T3SS）将数十种效应蛋白直接注射到宿主细胞的细胞质中。通过与同源伴侣蛋白的相互作用，III 型效应蛋白被定义并募集到分选平台，这是这些膜嵌入式纳米机器的细胞质成分。然而，值得注意的是，对文献的全面回顾表明，大多数 III 型效应蛋白的分泌尚未与伴侣蛋白相关联，这引发了关于未知伴侣蛋白的存在以及伴侣蛋白在效应蛋白分泌中的普遍性的问题。在这里，我们描述了第一个高通量 III 型分泌（T3S）测定法的开发，这是一种半自动固体板基测定法，可在多种条件下并排比较 20 多种效应蛋白的分泌。引人注目的是，我们发现大多数效应蛋白在野生型和不再编码一种或所有已知 III 型分泌效应蛋白伴侣蛋白的变体中以相当的水平分泌。此外，我们发现从表达核心 III 型分泌装置（T3SA）但没有其他特定蛋白的实验室菌株中有效地分泌了 III 型效应蛋白。此外，我们观察到定义伴侣蛋白依赖性和非依赖性效应蛋白所必需和足够的序列在本质上是不同的。总的来说，这些发现支持了一个主要的、以前未被认识到的、非典型的非伴侣蛋白独立分泌途径的存在，该途径可能对许多 T3SS 都是共同的。许多细菌病原体使用专门的纳米机器，包括 III 型分泌系统，将毒力蛋白（效应蛋白）直接注射到宿主细胞中。在这里，我们对 III 型效应蛋白分泌过程中的伴侣蛋白依赖性进行了首次广泛分析，为以前未被认识到的伴侣蛋白独立途径的存在提供了有力证据。这个非典型途径可能对许多细菌都很常见，因为对文献的广泛回顾表明，多个 III 型效应蛋白的分泌尚未与伴侣蛋白相关联。虽然还需要进一步的研究来辨别该途径的分子细节，但它的普遍性表明它可能成为开发抗菌剂的新目标。
