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標題: | 農桿菌第六型分泌系統效應子裝載機制的研究 Exploring the Effector Loading Mechanisms of a Type VI Secretion System in Agrobacterium tumefaciens |
作者: | Yun-Wei Lien 連允薇 |
指導教授: | 賴爾?(Erh-Min Lai) |
共同指導教授: | 洪挺軒 |
關鍵字: | 農桿菌,第六型分泌系統,VgrG,TssK,Tde,效應子裝載,蛋白質交互作用, Agrobacterium tumefaciens,type VI secretion system,VgrG,TssK,Tde,effector loading,protein-protein interaction, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 第六型分泌系統存在於許多革蘭氏陰性菌中,包括造成植物癌腫病的農桿菌。第六型分泌系統由四個構造組成,膜蛋白質複合體TssLM或TssJLM、底座Tss(A)EFGK、管狀構造Hcp與VgrG與可伸縮的外鞘TssBC。第六型分泌系統可以藉由分泌效應子到宿主細胞或是細菌競爭者中來提供病原性或提高適應性。農桿菌品系C58具有一個第六型分泌系統基因簇,包括imp操縱組與hcp操縱組,與一個位於染色體其他地方的孤兒vgrG2操縱組。農桿菌品系C58總共能產生三個效應子免疫蛋白質對,Tae-Tai、Tde1-Tdi1和Tde2-Tdi2。Tae被預測為一個肽聚醣酰胺酶,而Tde1和Tde2則為DNA分解酶。先前的研究顯示Tde1和Tde2會專一的由VgrG1和VgrG2運送,且Tde1需要Tap-1伴侶蛋白質,而Tde2需要Atu3641等伴侶蛋白質才能夠被分泌並維持其穩定性。然而,我們仍然不知道VgrG-Tde複合體如何被裝載至第六型分泌系統。VgrG和Hcp的分泌是第六型分泌系統功能性的一個重要指標。目前普遍認為非VgrG和Hcp的第六型分泌系統效應子並不是第六型分泌系統的一個零件,然而我們發現,刪除tde1-tdi1或tde2-tdi2將分別導致VgrG1或VgrG2的分泌消失。當tde1-tdi1和tde2-tdi2這兩個效應子免疫蛋白質基因對都被刪除時,Hcp的分泌將大量下降,而VgrG的分泌則完全無法被偵測。且tap-1突變株只有VgrG2的分泌,而atu3641突變株只能分泌VgrG1。這些發現讓我們假設第六型分泌系統效應子的裝載對VgrG與其他第六型分泌系統底座蛋白質的交互作用與組裝是重要的。接著,為了瞭解VgrG與其他底座蛋白質的交互作用,我們在大腸桿菌或是農桿菌中進行組胺酸標定(Histidine tag)的VgrG1的共純化實驗。在大腸桿菌中,我們發現VgrG1可以和TssA、TssF、TssG及TssK有交互作用,而與TssE沒有。而當Tap-1、Tde1和VgrG1-His與TssK一起表現在大腸桿菌中時,和沒有表現Tde1與Tap-1比起來,有更多的TssK被VgrG1-His共純化。在農桿菌中,組胺酸標定的VgrG1或VgrG2能夠共沉澱TssA與TssK,但無法共沉澱TssE。且組胺酸標定的TssK能夠共沉澱TssA、TssC、TssM與VgrG1-Tde1複合體。有趣的是,當兩個Tde都不存在時, TssK不再與TssM有交互作用。綜合以上的結果,我們推測只有有裝載Tde的VgrG能夠有效的和TssK進行交互作用,且TssK與TssM之間的交互作用需要這三個蛋白質形成的複合體;而第六型分泌系統只有在VgrG上有裝載Tde效應子時才能夠有效的發射。我們認為這可能是農桿菌或其他具有第六型分泌系統的細菌在細胞內沒有效應子蛋白質時所做出的節省能量的策略。
除了藉由VgrG來運送的Tde,我們發現藉由Hcp運送的Tae可能具有與Tde不同的調控第六型分泌系統的方式。我們發現,刪除tae的基因將會導致tde-tdi雙突變株的Hcp分泌量上升。這有可能是因為Tae可以扮演類似噬菌體中的tape measure protein或是capping protein的功能。這兩個蛋白質能夠控制噬菌體的鞘的長度或是防止管狀構造在伸縮的時候滑出。 Type VI secretion system (T6SS) is a molecular machinery widespread in Gram-negative bacteria including the causal agent of plant crown gall disease, Agrobacterium tumefaciens. A T6SS machine is composed of a membrane complex [Tss(J)LM], a baseplate [Tss(A)EFGK], and a contractile tube consisting an Hcp-VgrG inner tube wrapped by an outer TssBC sheath. This nanomachine has been deployed to deliver diverse effector proteins into eukaryotic host cells or bacterial competitors to increase the fitness of T6SS-possessing bacteria. A. tumefaciens strain C58 encodes one main T6SS gene cluster comprising imp operon and hcp operon as well as one orphan vgrG2 operon encoded elsewhere. Three toxin-immunity pairs (Tae-Tai, Tde1-Tdi1 and Tde2-Tdi2) were produced. Among them, Tde1 and Tde2 are DNase toxins and Tae is a putative peptidoglycan amidase. These Tde DNase effectors are delivered by a VgrG-specific manner, in which Tde1 and Tde2 are cargo effectors of VgrG1 and VgrG2, respectively. Chaperone/adaptor proteins are also involved in effector loading to VgrG because Tap-1 interacts with Tde1 for binding to VgrG1 tip and Atu3641 is required for VgrG2-mediated Tde2 delivery. Nevertheless, how this effector-VgrG complex is loaded onto a T6SS machine for secretion still remains unknown. Secretion of Hcp tube and VgrG spike is regarded as a hallmark of T6SS firing. Though it is widely believed that cargo effectors (or non-VgrG/Hcp effectors) are not a component of a Type IV secretion apparatus, we found that deletion of tde1-tdi1 or tde2-tdi2 diminishes VgrG1 and VgrG2 secretion, respectively. Furthermore, VgrG1 secretion is abolished in Δtap-1 whereas VgrG2 is no longer secreted in Δatu3641. These findings suggest that Tde effector loaded onto its cognate VgrG is required for assembly of a functional T6SS for secretion. Next, I determined how VgrG-effector complex is loaded onto membrane-associated T6SS subcomplex for T6SS firing. To test whether VgrG and VgrG-Tde complex interact with baseplate components, protein-protein interaction studies were carried out by co-purification in Escherichia coli and in A. tumefaciens. E. coli co-purification assay results showed that among the baseplate proteins, VgrG interacts with TssA, TssF, TssG and TssK but not TssE. Higher levels of VgrG1-TssK interactions were detected when co-expression of Tap-1, Tde1, Tdi1 and PAAR in E. coli, suggesting that Tde1 loaded onto VgrG1 enhances VgrG1 interaction to TssK. In A. tumefaciens, TssA and TssK but not TssE are co-purified with His-tagged VgrG1 or VgrG2 proteins. On the other hand, His-tagged TssK is able to co-purify TssA, TssC, TssM and VgrG1-Tde1 complex in wild type A. tumefaciens. Interestingly, TssK no longer interacts with TssM in the absence of any Tde effectors, suggesting that Tde loading onto its cognate VgrG is important for TssK-TssM interaction. Taken together, we suggest that only Tde-loaded VgrG can bind strongly to baseplate component TssK and such tripartite interaction is required for TssK recruitment to TssM-TssL membrane complex for initiation of T6SS assembly and firing. We propose that this is a strategy utilized by A. tumefaciens and likely also other T6SS-possessing bacteria to save energy when VgrG cargo effector is not loaded. Other than VgrG cargo effector Tde, Tae that is likely to be the Hcp cargo effector was found to regulate T6SS through a different way. In acidic minimal medium, deletion of tae locus results in Hcp secretion restoration in tde-tdi double deletion mutant. It is possible that beside being a bacterial toxin, Tae may also function as a protein controlling the length of Hcp tube and TssBC sheath or preventing Hcp tube from sliding out during contraction, similar to tape measure protein or capping protein of bacteriophage. |
URI: | http://tdr.lib.ntu.edu.tw/handle/123456789/1136 |
DOI: | 10.6342/NTU201802417 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 植物病理與微生物學系 |
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