Deciphering the functions of type II and type IV secretion systems from the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria

革兰氏阴性植物致病菌油菜黄单胞菌。辣椒、番茄花斑病菌是辣椒、番茄细菌性斑疹病的病原，是研究细菌毒力因子分泌的模式生物之一。Xcv致病性的关键是III型分泌系统，它将细菌效应蛋白转运到真核细胞中。此外，Xcv与寄主植物的有效相互作用依赖于xps - II型分泌（T2S）系统，该系统将降解酶分泌到细胞外环境。我们之前的研究表明，不同病原菌的T2S分泌组不同，这与之前预期的细菌T2S底物特异性的保守性相反。Xcv的Xps-T2S系统分泌蛋白酶、木聚糖酶和脂肪酶，这些酶有助于细菌的毒力。在缺乏功能性T2S系统的情况下，Xcv的T2S底物分泌并未完全消除，这表明它们是针对其他运输途径的。与这一观察结果一致，我们在Xcv的外膜囊泡（OMVs）中检测到T2S底物。在本项目中，我们想要描述omv对Xcv毒力因子分泌的贡献。此外，我们建议研究T2S底物与分泌器官成分之间可能的相互作用，并确定参与底物对接的蛋白质区域。除了分析T2S系统和omv外，我们还对Xcv的两种IV型分泌系统（T4S）进行了功能表征，这两种系统分别类似于农杆菌的VirB/D4-T4S系统和嗜肺军团菌的Icm/Dot-T4S系统。Xcv是唯一已知的植物致病菌，它含有Icm/ dot样T4S系统。我们最近的突变体研究表明，这两个T4S系统都参与了油菜葡萄球菌pv的相互作用。易受感染的辣椒植株。此外，生物信息学方法鉴定了候选T4S底物，这些底物与已知的T4S效应物同源和/或含有保守的c端氨基酸基序，可能作为分泌信号。在目前的项目中，我们将研究Xcv候选T4S底物的分泌和易位及其对细菌毒力的潜在贡献。此外，鉴于最近的报道，VirB/ d4样T4S系统的作用，从相关病原体轴索黄单胞菌pv。我们将研究T4S系统在Xcv与其他细菌细胞相互作用中的可能作用。

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease on pepper and tomato plants and one of the model organisms to study secretion of bacterial virulence factors. Essential for pathogenicity of Xcv is the type III secretion system, which translocates bacterial effector proteins into eukaryotic cells. Furthermore, the efficient interaction of Xcv with its host plants depends on the Xps-type II secretion (T2S) system, which secretes degradative enzymes into the extracellular milieu. We previously showed that the T2S secretomes vary in different pathovars of X. campestris, which is in contrast to the previously anticipated conservation of bacterial T2S substrate specificities. The Xps-T2S system from Xcv secretes proteases, xylanases and a lipase, which contribute to bacterial virulence. In the absence of functional T2S systems, secretion of T2S substrates from Xcv was not completely abolished, suggesting that they are targeted to alternative transport routes. In agreement with this observation, we detected T2S substrates in outer membrane vesicles (OMVs) of Xcv. In the present project, we want to characterize the contribution of OMVs to the secretion of virulence factors from Xcv. Furthermore, we propose to investigate possible interactions between T2S substrates and components of the secretion apparatus and to identify protein regions, which are involved in substrate docking. In addition to the analysis of the T2S system and OMVs, we aim at the functional characterization of the two type IV secretion (T4S) systems of Xcv, which resemble the VirB/D4-T4S system from Agrobacterium tumefaciens and the Icm/Dot-T4S system from the animal-pathogenic bacterium Legionella pneumophila, respectively. Xcv is the only known plant-pathogenic bacterium, which contains an Icm/Dot-like T4S system. Our recent mutant studies revealed that both T4S systems contribute to the interaction of X. campestris pv. vesicatoria with susceptible pepper plants. Furthermore, bioinformatic approaches led to the identification of candidate T4S substrates, which are homologous to known T4S effectors and/or contain conserved C-terminal amino acid motifs, which presumably serve as secretion signals. In the present project, we will study secretion and translocation of candidate T4S substrates from Xcv and their potential contribution to bacterial virulence. Furthermore, given the recent report on the role of the VirB/D4-like T4S system from the related pathogen Xanthomonas axonopodis pv. citri in bacterial killing, we will investigate a possible role of T4S systems in the interaction of Xcv with other bacterial cells.