PDF(Pubmed)
Abstract：
Many Gram-negative pathogens rely on type IV secretion systems (T4SS) for infection. One limitation has been the lack of ideal reporters to identify T4SS translocated effectors and study T4SS function. Most reporter systems make use of fusions to reporter proteins, in particular, β-lactamase (TEM) and calmodulin-dependent adenylate cyclase (CYA), that allow detection of translocated enzymatic activity inside host cells. However, both systems require costly reagents and use complex, multistep procedures for loading host cells with substrate (TEM) or for analysis (CYA). Therefore, we have developed and characterized a novel reporter system using nanoluciferase (NLuc) fusions to address these limitations. Serendipitously, we discovered that Nluc itself is efficiently translocated by Legionella pneumophila T4SS in an IcmSW chaperone-dependent manner via an N-terminal translocation signal. Extensive mutagenesis in the NLuc N terminus suggested the importance of an α-helical domain spanning D5 to V9, as mutations predicted to disrupt this structure, with one exception, were translocation defective. Notably, NLuc was capable of translocating several proteins examined when fused to the N or C terminus, while maintaining robust luciferase activity. In particular, it delivered the split GFP11 fragment into J774 macrophages transfected with GFPopt, thereby resulting in in vivo assembly of superfolder green fluorescent protein (GFP). This provided a bifunctional assay in which translocation could be assayed by fluorescence microplate, confocal microscopy, and/or luciferase assays. We further identified an optimal NLuc substrate which allowed a robust, inexpensive, one-step, high-throughput screening assay to identify T4SS translocation substrates and inhibitors. Taken together, these results indicate that NLuc provides both new insight into and also tools for studying T4SS biology. IMPORTANCE Type IV secretion systems (T4SS) are used by Gram-negative pathogens to coopt host cell function. However, the translocation signals recognized by T4SS are not fully explained by primary amino acid sequence, suggesting yet-to-be-defined contributions of secondary and tertiary structure. Here, we unexpectedly identified nanoluciferase (NLuc) as an efficient IcmSW-dependent translocated T4SS substrate, and we provide extensive mutagenesis data suggesting that the first N-terminal, alpha-helix domain is a critical translocation recognition motif. Notably, most existing reporter systems for studying translocated proteins make use of fusions to reporters to permit detection of translocated enzymatic activity inside the host cell. However, existing systems require extremely costly substrates, complex technical procedures to isolate eukaryotic cytoplasm for analysis, and/or are insensitive. Importantly, we found that NLuc provides a powerful, cost-effective new tool to address these limitations and facilitate high-throughput exploration of secretion system biology.
摘要：
许多革兰氏阴性病原体依赖于IV型分泌系统(T4SS)进行感染。一个限制是缺乏鉴定T4SS易位效应子和研究T4SS功能的理想报告基因。大多数报告系统利用融合来报告蛋白质，特别是,β-内酰胺酶（TEM）和钙调蛋白依赖性腺苷酸环化酶（CYA），允许检测宿主细胞内易位的酶活性。然而,这两个系统都需要昂贵的试剂，并且使用复杂的，用底物(TEM)装载宿主细胞或用于分析(CYA)的多步骤程序。因此,为了解决这些限制,我们开发并表征了使用纳米荧光素酶(NLuc)融合体的新型报告系统.偶然,我们发现Nluc本身通过N末端易位信号以IcmSW伴侣依赖性方式被嗜肺军团菌T4SS有效易位。NLucN末端的大量诱变表明了跨越D5至V9的α-螺旋结构域的重要性，因为预测突变会破坏该结构，除了一个例外,有易位缺陷。值得注意的是,当融合到N或C末端时，NLuc能够易位检查的几种蛋白质，同时保持强大的荧光素酶活性。特别是,它将分裂的GFP11片段传递到用GFPopt转染的J774巨噬细胞中，从而导致超折叠绿色荧光蛋白(GFP)的体内组装。这提供了一种双功能测定，其中易位可以通过荧光微孔板进行测定，共聚焦显微镜，和/或荧光素酶测定。我们进一步确定了一个最佳的NLuc底物，它允许一个强大的，便宜,一步,高通量筛选试验鉴定T4SS易位底物和抑制剂。一起来看,这些结果表明,NLuc为研究T4SS生物学提供了新的见解和工具.重要性IV型分泌系统（T4SS）被革兰氏阴性病原体用来调节宿主细胞功能。然而,T4SS识别的易位信号不能完全由一级氨基酸序列解释，表明二级和三级结构的贡献尚未确定。这里,我们意外地确定了纳米荧光素酶（NLuc）作为一种有效的IcmSW依赖性易位T4SS底物，我们提供了大量的诱变数据，表明第一个N端，α-螺旋结构域是关键的易位识别基序。值得注意的是,大多数用于研究易位蛋白质的现有报告系统都利用与报告分子的融合来允许检测宿主细胞内易位的酶活性。然而,现有的系统需要极其昂贵的基板，分离真核细胞质进行分析的复杂技术程序，和/或不敏感。重要的是,我们发现NLuc提供了一个强大的，具有成本效益的新工具来解决这些限制，并促进分泌系统生物学的高通量探索。
