第六型分泌系統在 Azorhizobium 與 Agrobacterium 之功能與機制研究

Abstract

第六型細菌分泌系統（Type VI secretion system, T6SS）是一個由13-14個核心蛋白質所組成、形狀類似噬菌體尾部、結構橫跨革蘭氏陰性細菌內外雙膜的蛋白質外泌系統，可藉由組裝成長管柱構造穿透真核生物、或原核生物等受體細胞（recipient cells）的細胞膜，將效應蛋白質注入至受體細胞中。綜合許多研究發現，T6SS的功能除了與致病力有關外，也與細菌間競爭、抗真核生物活性、金屬離子螯合、促進基因水平轉移等息息相關。雖然T6SS已知存在四分之一以上的革蘭氏陰性細菌基因體中，其中有許多是植物相關細菌（plant-associated bacteria），包括了病原菌及共生菌，然而T6SS在這些細菌中所扮演的生物功能至今仍然存在許多未知。有鑑於此，本研究使用共生莖瘤固氮根瘤菌（Azorhizobium caulinodans）與致病根癌農桿菌（Agrobacterium tumefaciens）作為材料，闡明T6SS在植物共生菌與植物病原菌上所扮演的角色、功能與機制。 A. caulinodans ORS571基因體上有一套T6SS基因簇，本研究依此建構了一系列T6SS核心蛋白基因缺失突變株，用以探討T6SS分別對於菌株在自由態與共生態上的影響。研究結果發現，雖然有無T6SS對於自由態ORS571菌株的生長、菌體形態、自由態固氮能力、細菌間競爭、在植物上的定植能力以及共生效益並無顯著影響，當缺失T6SS基因的ORS571菌株與豆科宿主長喙田菁（Sesbania rostrata）建立共生關係時，其共生競爭能力與野生株相較顯著較低。 根癌農桿菌（A. tumefaciens）是許多癌腫病的病原菌，其T6SS是一種能夠殺死種間和種內細菌的抗菌武器，故本研究使用 A. tumefaciens T6SS來研究細菌間競爭的作用模式。儘管其T6SS的分子調控與結構組織已被廣泛研究，但對於受攻擊方的受體細胞（recipient cell）因子所知甚少。因此本研究以A. tumefaciens strain C58為攻擊者，而缺乏T6SS的大腸桿菌為受體細胞，建立了一個高通量細菌競爭篩選平台，以尋找對T6SS殺滅力具有抗性的受體菌株。經由篩選得到16株對C58菌株的 T6SS殺滅力呈現低敏感表型的突變株，並確認其中四個與增強C58菌株的T6SS殺滅力有關的大腸桿菌基因（clpP, gltA, ydhS, ydaE）。進一步探討ClpP蛋白酶與T6SS生物功能關連性的試驗結果顯示，當ClpP蛋白酶與其銜接蛋白ClpA形成ClpAP複合物時，大腸桿菌對根癌農桿菌C58菌株的T6SS攻擊的敏感性便會顯著提昇。 綜合以上結果，本研究指出共生根瘤菌A. caulinodans ORS571可藉由T6SS確保其共生競爭性，進而提昇結瘤佔有率；病原菌A. tumefaciens strain C58的細菌間競爭則需要多個受體因子的參與，來達到最佳的T6SS殺滅力。這些發現不僅擴展了我們對植物相關細菌T6SS的理解，且所獲得的知識或可應用於其他具有T6SS細菌的相關研究上。

The type VI secretion system (T6SS) is a protein secretion system composed of 13-14 core proteins, which structurally resembles an inverse phage tail. The T6SS spans the inner and outer membranes of a Gram-negative bacterium and can penetrate the membranes of the eukaryotic or prokaryotic recipient cells. The biological functions of T6SS are versatile, ranging from anti-eukaryotic activity, virulence, interbacterial competition, metal ion sequestering, and facilitating horizontal gene transfer, many of which display in animal-associated bacteria. Despite the existence in more than a quarter of the sequenced Gram-negative bacteria, the biological functions of T6SS in plant-associated bacteria, including mutualistic and pathogenic bacteria, remained mostly unknown. This study used a mutualistic bacterium, Azorbizobium caulinodans, and a pathogenic bacterium, Agrobacterium tumefaciens, to elucidate the functional and mechanistic aspects of T6SS in rhizobacteria. With the discovery of a T6SS gene cluster in A. caulinodans ORS571, a series of T6SS gene deletion mutants were generated, and their phenotypes were analyzed in free-living and symbiotic states. The results showed that whether the T6SS exists or not, there was no detectable effect on vegetative growth, morphology, free-living nitrogen-fixing ability, interbacterial competition, plant colonization, or symbiotic effectiveness. On the other hand, the strains lacking T6SS showed a reduction in the symbiotic competitiveness when co-infected with a wild-type strain on the stem of the leguminous host plant Sesbania rostrata. A. tumefaciens is a causative agent of crown gall disease in a wide range of plants and harbors T6SS. The T6SS of A. tumefaciens strain C58 is an antibacterial weapon capable of killing both inter- and intra-species bacteria. Thus, A. tumefaciens C58 is selected to study the mode of action of T6SS during interbacterial competition. While the molecular mechanisms and structural organization of the T6SS have been extensively studied, little was known about the recipient cell factor(s) needed or subverted for an attack to be successful. Thus, a high-throughput interbacterial competition screening platform was established in search of the recipient strains that were resistant to T6SS killing. A. tumefaciens strain C58 served as a model attacker, and the Escherichia coli devoid of T6SS served as a recipient cell. From the screening, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified. Among them, four genes (clpP, gltA, ydhS, ydaE) that participate in enhancing the recipient susceptibility to A. tumefaciens T6SS killing were confirmed. Further studies demonstrated that the ClpP protease and its adapter protein ClpA forming the ClpAP complex in the E. coli recipient cell act in enhancing the recipient’s susceptibility to the A. tumefaciens T6SS attack. In summary, this study discovered that the T6SS of legume symbiont A. caulinodans ORS571 functions to ensure the competitiveness of nodule occupancy and that multiple recipient factors are required to maximize T6SS killing efficiency of A. tumefaciens C58. The findings expand our understanding of T6SS in plant-associated bacteria, and the knowledge obtained could be applied to other T6SS harboring bacteria.