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细菌可以引起植物、动物以及人类严重的病害,在其侵染过程中,主要通过多种分泌系统以完成与宿主之间的互作[1-2]。其中,Ⅵ型分泌系统(T6SS, Type VI secretory system)可以通过释放效应蛋白至邻近靶细胞,包括对于宿主的攻击性和种间竞争的防御性等作用[3]。此外,T6SS还具有参与生物被膜形成等能力[4],在细菌的营养竞争、生存定殖、侵染宿主致病等过程中发挥着重要作用[5]。
有关T6SS的研究可以追溯至1996年,当时,学者对霍乱弧菌(Vibrio cholerae)中一个缺乏可识别信号肽的序列HCP(Hemolysin Coregulated Protein)横跨细胞内外膜分泌蛋白开展研究,明确其对病原细菌的毒力、定殖能力以及环境适应性等有重要影响[6]。2006年,Pukatzki等人发现除HCP外,V. cholerae中的VgrG蛋白同样存在胞外输出现象,然而,其均不含信号肽且与T3SS或T4SS并不相同,因此,首次提出了T6SS概念[7]。此后,2012年《Nature》论文系统阐述了T6SS分泌模型,即一个通过独特的基座结构与内膜相连,往复于延伸或收缩构象的长管外包弹簧状结构鞘(主要由HCP、TssB/C组成),保持在组装、快速收缩、释放、拆卸和重新组装之间循环[8],与T4噬菌体类似,基座结构将收缩信号传递给鞘,并且拥有保守的触发机制[9]。通常细菌基于T6SS系统以接触依赖性传输毒性蛋白,诸如肽聚糖酶[10]、磷脂酶[11]、ATP酶等效应子[12],面对营养压力时通过T6SS将其输送至竞争细胞,从而导致靶细胞死亡。
学术界关于细菌中T6SS的研究多以铜绿假单胞菌(Pseudomonas aeruginosa)为主[13],而有关植物病原细菌T6SS的研究,最早见于2010年关于梨火疫病菌(Erwinnia amylovora)[14]的报道。鉴于丁香假单胞菌(Pseudomonas syringae)[15]、青枯雷尔氏菌(Ralstonia solanacearum)[16]、根癌农杆菌(Agrobacterium tumefaciens)[17]、梨火疫病菌[18]、稻生黄单胞菌(Xanthomonas oryzae pv. oryzicola, Xoc)[19]等5种植物病原细菌对农林业生产危害的严重性,有关上述细菌T6SS的调控机制[20]、作用机制[18]以及结构功能[21]等研究报道逐年增加。然而,尚未见不同植物病原细菌中T6SS的系统性对比分析报道。因此,本研究以前人研究成果为基础,对上述植物病原细菌中T6SS蛋白的数量、种类、遗传分类以及结构功能进行综述,以期明确不同植物病原细菌中T6SS在其功能蛋白及遗传关系等方面存在的差异性,并指出了T6SS研究中存在的一些问题。
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T6SS主要存在于革兰氏阴性细菌中[6,22],以P. aeruginosa为例,典型的T6SS装配结构与噬菌体尾部穿膜结构类似[23-24],存在于内外膜组织之间,是基于细胞接触依赖性的多组件跨膜通道。笔者利用美国国立卫生院(NCBI)数据库,对5种植物病原细菌(P. syringae、R. solanacearum、A. tumefaciens、E. amylovora、Xoc;登录号分别为:GCF_016694755.2、GCF_001587155.1、GCF_003667905.1、GCF_000091565.1、GCF_001021915.1)中共138个T6SS蛋白序列进行搜索下载、整理,利用蛋白保守结构域分析软件SMART[25]进行预测分析,发现19种T6SS蛋白存在于5种细菌中,且不同种细菌拥有蛋白的类别与数量不尽相同,具有一定多样性。同时,除VgrG的蛋白序列长度在不同细菌之间差异较大,为296~917 aa,其余同种T6SS蛋白之间长度差别相对较小,具有一定保守性(图1)。T6SS蛋白种类主要包括跨膜复合体TssM(ImcF)、TssJ(SciN)、TssL(DotU);基座亚基TssE(GPW_gp25)、TssF、TssG、TssK(VasE);弹簧状伸缩鞘大亚基TssC(VipB)、小亚基TssB(VipA)、管状蛋白HCP;尖端效应子结合蛋白VgrG(Phage_GPD);装卸辅助蛋白TssA(ImpA_N);调控蛋白TagF(DUF2094)、TagH(FHA)、TssH(Clp_N);酰胺酶效应蛋白Tae4、酰胺酶免疫蛋白Tai4。此外,E. amylovora与Xoc分别独有IcmF(Vask)、CagE_TrbE_VirB等结构域的T6SS分泌蛋白(蛋白ID分别为WP_004160264.1、WP_047340360.1)。通过对T6SS蛋白结构域进行解析,明确不同病原细菌具有相似的T6SS蛋白组成、不同的蛋白数量以及部分物种特有的结构域,与前人明确R. solanacearum中HCP、VgrG蛋白的三级结构分别与P. aeruginosa、大肠杆菌(Escherichia coli)的HCP、VgrG相似等结果类似[26]。
图 1 T6SS在5种不同植物病原细菌中的相关蛋白保守结构域种类情况
Figure 1. Conserved domains of T6SS related proteins in five different plant pathogenic bacteria
具体而言,VgrG是一个三聚体状的尖端效应子结合蛋白,是对靶细胞执行毒性功能的核心蛋白[27],在5种植物病原细菌中均具有较多的数量(表1);TssM/J/L是一个跨内外膜复合体结构,是效应子等蛋白的膜运输通道[28];TssE/F/G/K是一个锚定于内膜并链接TssM/J/L结构的漏斗状复合体基座,用以VgrG蛋白的识别与吸附[29];HCP是一个管状蛋白,当T6SS开始装配时,其一端依附于VgrG,行使VgrG等尖端蛋白受力功能[16];TssB/C是一个空心弹簧状蛋白复合体,包裹于HCP外侧,是VgrG与HCP等结构被执行推力的来源[28];TssA是一个T6SS装卸辅助识别蛋白,当T6SS开始装配时,其一侧可分别粘连VgrG、TssB/C和HCP,并有序进行组装[30];TssH属于AAA + ATP酶超家族和ClpV亚家族,主要在TssB/C鞘收缩释放T6SS效应子过程中起作用[31];TagF通过与TagH相互作用来调节T6SS效应子的分泌过程[32];Tae4作为效应子参与细菌竞争中对目标细菌肽聚糖和膜脂的侵蚀;Tai4作为免疫蛋白用于抵御Tae4,它们与VgrG基因编码相关联。上述蛋白具有不同的功能,使T6SS在不同环境下执行多种生存策略,分泌多种效应蛋白,从而在结构域之间以及结构域与效应子之间具有相互紧密配合的特点,进而适应多变的细胞环境。
表 1 5种不同植物病原细菌所包含T6SS相关蛋白情况对比
Table 1. Comparison of T6SS related proteins in five different plant pathogenic bacteria
蛋白 Protein P. syringae R. solanacearum A. tumefaciens E. amylovora Xoc 合计 Total VgrG 6 6 1 5 2 20 TssM 2 1 1 2 2 8 TssJ 2 1 0 3 1 7 TssL 0 0 1 2 0 3 TssE 2 1 1 2 2 8 TssF 2 1 1 2 2 8 TssG 2 1 1 2 2 8 TssK 2 1 1 2 2 8 HCP 7 1 1 2 2 13 TssB 2 1 1 2 2 8 TssC 2 1 2 2 2 9 TssA 2 1 1 3 2 9 TssH 2 1 1 2 2 8 TagF 1 1 1 2 2 7 TagH 0 0 0 2 1 3 Tae4 1 1 1 1 0 4 Tai4 2 1 0 2 0 5 VasK SP 0 0 0 1 0 1 CagE SP 0 0 0 0 1 1 合计 Total 37 20 15 39 27 138 一般而言,T6SS中的TssM/J/L、TssE/F/G/K分别被组装成跨膜通道和基座复合体[8],随后TssB/C弹簧鞘的收缩推动HCP管、VgrG突刺和效应子并通过T6SS跨膜通道离开细胞,进入细胞外环境或附近的另一个细胞[33](图2),毒性效应子发挥作用从而导致靶细胞生长抑制或死亡[10-11]。
植物病原细菌Ⅵ型分泌系统研究进展
A Review on the Type Ⅵ Secretion System of Plant Pathogenic Cacteria
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摘要:
目的 Ⅵ型分泌系统(T6SS)作为植物病原细菌发挥重要功能的调控系统之一,主要表现为通过效应子的释放对宿主植物的攻击性和自身在种间竞争过程中面对胁迫反应的防御性等方面,明确重要植物病原细菌的T6SS蛋白及功能差异,有助于推动预防和治疗植物病害的新型农药的开发和利用。 方法 通过对比分析5种重要植物病原细菌(丁香假单胞菌、青枯雷尔氏菌、根癌农杆菌、梨火疫病菌、稻生黄单胞菌)中138个T6SS蛋白的保守结构域以及遗传关系,同时,基于对国内外文献数据库中有关上述5种细菌的T6SS功能研究的文献开展分析。 结果 明确5种植物病原细菌中T6SS蛋白在种类和数量上存在差异性。根据蛋白序列同源性及结构域,上述蛋白分为3大类,其中,含有VgrG、HCP等结构域的T6SS蛋白具有很好的聚类区分度,而含有TssA、TssG等结构域的T6SS蛋白则可分散在不同类别中。 结论 明确5种植物病原细菌中T6SS蛋白及功能具有一定保守性和独特性特征。该研究为进一步开展不同植物病原细菌中T6SS蛋白的预测分析,以及不同T6SS蛋白之间的关系解析提供理论基础。 Abstract:Objective Type Ⅵ secretion system (T6SS) is one of the regulatory systems that plays important functions in plant pathogenic bacteria. In terms of defense and other aspects, clarifying the T6SS protein and functional differences of important plant pathogenic bacteria will help promote the development and utilization of new pesticides for the prevention and treatment of plant diseases. Method In the study, five kinds of important plant pathogenic bacteria (Pseudomonas syringae, Ralstonia solanacearum, Agrobacterium tumefaciens, Erwinnia amylovora and Xanthomonas oryzae pv. oryzicola) bacterium in 138 conservative T6SS proteins structure domain and genetic relationships were analyzed, and the literatures about the above five kinds of bacteria in the database of the T6SS function research were also analyzed. Results T6SS proteins in the five kinds of plant pathogenic bacteria were different in species and quantity. According to proteins sequence homology and domain, the above proteins could be divided into three categories. Among them, T6SS proteins containing VgrG, HCP and other domains had good clustering discrimination, while T6SS proteins containing TssA, TssG and other domains could be divided into different categories. Conclusion The proteins and function of T6SS in the five kinds of plant pathogenic bacteria have certain conservation and unique characteristics. This study provides a theoretical basis for further prediction and analysis of T6SS proteins in different plant pathogenic bacteria and the relationship between different T6SS proteins. -
Key words:
- Plant pathogens bacteria
- / T6SS
- / Structural domain
- / Function
- / Genetic relationships
- / review
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表 1 5种不同植物病原细菌所包含T6SS相关蛋白情况对比
Table 1. Comparison of T6SS related proteins in five different plant pathogenic bacteria
蛋白 Protein P. syringae R. solanacearum A. tumefaciens E. amylovora Xoc 合计 Total VgrG 6 6 1 5 2 20 TssM 2 1 1 2 2 8 TssJ 2 1 0 3 1 7 TssL 0 0 1 2 0 3 TssE 2 1 1 2 2 8 TssF 2 1 1 2 2 8 TssG 2 1 1 2 2 8 TssK 2 1 1 2 2 8 HCP 7 1 1 2 2 13 TssB 2 1 1 2 2 8 TssC 2 1 2 2 2 9 TssA 2 1 1 3 2 9 TssH 2 1 1 2 2 8 TagF 1 1 1 2 2 7 TagH 0 0 0 2 1 3 Tae4 1 1 1 1 0 4 Tai4 2 1 0 2 0 5 VasK SP 0 0 0 1 0 1 CagE SP 0 0 0 0 1 1 合计 Total 37 20 15 39 27 138 -
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