Type VI secretion in Pseudomonas species: bacterial competition and biocontrol

铜绿假单胞菌是一种革兰氏阴性细菌，在土壤、潮湿的表面或植物等多种环境中茁壮成长。它有能力成为一种成功的病原体，并感染广泛的宿主，如阿米巴、蠕虫、昆虫、植物或动物。它也是一种可怕的人类病原体，众所周知，囊性纤维化患者的肺部会发生无法治愈的感染。由于环境和宿主的多样性，铜绿假单胞菌遇到了大量的微生物，它们定居在相同的生态位上，争夺相同的资源。研究发现，一种名为VI型分泌系统(T6SS)的分子武器在由此引发的细菌战中发挥了重要作用。T6SS类似于噬菌体尾巴，但它不是将DNA注入猎物细胞，而是注入细菌毒素。这些毒素会降解基本的细菌成分，如细胞壁/包膜或DNA，从而导致不可逆转的损害和细胞死亡。铜绿假单胞菌在T6SS的研究中发挥了重要作用，在该领域的研究中取得了许多突破。T6SS依赖杀灭试验表明，T6SS对大多数革兰氏阴性猎物具有较强的杀灭作用。杀灭试验允许监测存放在琼脂平板上的一滴共培养的幸存猎物细胞。铜绿假单胞菌对T6SS的攻击不是系统性的，通常发生在遇到的生物具有威胁性的时候，例如通过展示T6SS武器库，进行决斗。T6SS毒素的数量比预期的要全面得多。许多T6SS毒素具有甚至无法预测的功能。然而，将这些分子注射到目标细菌中会导致死亡，这表明新的抗菌活性有待发现。我们开始在铜绿假单胞菌中展开T6SS毒素谱，发现其他假单胞菌都有活性的T6SS和独特的T6SS毒素谱。这就是植物病原体丁香假单胞菌或植物有益生物体恶臭假单胞菌的情况。在体外杀伤试验中，三种假单胞菌均优于大肠埃希菌，而铜绿假单胞菌优于恶臭假单胞菌和丁香假单胞菌。我们发现，即使铜绿假单胞菌在体外胜过植物病原菌根癌农杆菌，但在植物中共接种后，这些微生物之间的权力关系发生了变化，根癌农杆菌占优势。这些研究揭示了一种复杂的细菌间关系，其中T6SS是核心的，但受到生物体之间相互作用的特殊性以及它们聚集的条件和环境的影响。在我们的项目中，我们调查了T6SS如何影响细菌种群的进化和多微生物群落的组成。我们希望在我们的专业知识和开创性工作的基础上，识别新的T6SS毒素，如DNase TDE，假单胞菌中T6SS的特征，以及T6SS熟练掌握的生物之间的植物竞争分析的使用。我们想要监测混合细菌种群在动态环境中的演变，例如使用流动细胞和荧光标记生物的混合生物膜。生物膜是复杂细菌种群的一种自然生活方式。每株细菌携带不同的T6SS谱系，从而了解单个T6SS特征在生物膜组成结果中的作用/重要性。总之，我们将使用不同的环境设置，即体外、混合生物膜和植物分析，研究T6SS在不同假单胞菌物种中的作用。所获得的知识应该有助于表征新的抗菌活性，了解细菌种群的进化，以及为控制作物病害而设计对植物有益的恶臭疫霉菌株。这项研究将在生态学和农业(根部定植)、医学(抗菌)和基础科学等领域产生影响。

Pseudomonas aeruginosa is a gram-negative bacterium that thrives in a multitude of environmental niches, e.g. soil, moist surfaces or plants. It has the ability to become a successful pathogen and infects a wide range of hosts, e.g. amoeba, worms, insects, plants or animals. It is also a dreadful human pathogen and is known for incurable infections in the lungs of cystic fibrosis patients. Because of this versatility of environments and hosts, P. aeruginosa encounters numerous microorganisms colonizing the same niche and competing for the same resources. It was found that a molecular weapon, called the type VI secretion system (T6SS), plays an important part in the resulting bacterial warfare.The T6SS is akin a bacteriophage tail, but instead of injecting DNA in prey cells, it injects bacterial toxins. These toxins degrade essential bacterial components, such as the cell wall/envelope or DNA, which results in irreversible damages and cell death. P. aeruginosa has been instrumental in T6SS research and numbers of breakthroughs in the field were made upon studying this organism. The development of T6SS-dependent killing assay showed that the P. aeruginosa T6SS is potent and kills most gram-negative preys. The killing assay allows the monitoring of surviving prey cells in a drop of co-culture deposited on an agar plate. The T6SS attack by P. aeruginosa is not systematic and often occurs when the encountered organism is threatening, for example by exhibiting a T6SS armory, which engages the duel.The number of T6SS toxins is far more comprehensive than anticipated. Many T6SS toxins have function that could not even be predicted. Yet the injection of these molecules in target bacteria results in death, suggesting that new antibacterial activities are to be found. We begun to unfold the T6SS toxin repertoire in P. aeruginosa and discovered that other Pseudomonas species have an active T6SS and a unique T6SS toxins repertoire. This is the case for Pseudomonas syringae, a plant pathogen, or Pseudomonas putida, a plant beneficial organism. All three Pseudomonas outcompete Escherichia coli in the in vitro killing assay, while P. aeruginosa outcompetes both P. putida and P. syringae. We showed that even though P. aeruginosa outcompetes the plant pathogen Agrobacterium tumefaciens in vitro, upon co-inoculation in planta the power relations in between these organisms is changed and A. tumefaciens prevails. These studies revealed a complex inter-bacterial relationship, in which the T6SS is central but is influenced by the specificity of interactions between organisms and the conditions and environments in which they met.In our project we investigate how the T6SS influences evolution of bacterial population and composition of polymicrobial communities. We want to build on our expertise and pioneering work in the identification of new T6SS toxins such as the DNase Tde, the characterization of T6SSs in Pseudomonas species and the use of in planta competition assays between T6SS-proficient organisms. We want to monitor the evolution of mixed bacterial populations in a dynamic environment, such as mixed biofilms using flow cells and fluorescent-tagged organisms. Biofilm is a natural lifestyle of complex bacterial populations. Each bacterial strain carries a distinct T6SS repertoire thus giving access to the role/importance of individual T6SS feature in the outcome of biofilm composition.In conclusion, we will investigate the role of the T6SS in distinct Pseudomonas species using various environmental set-ups, i.e. in vitro, mixed biofilms and in planta assays. The knowledge acquired should result in the characterization of novel antibacterial activities, understanding in the evolution of bacterial population and engineering of plant beneficial P. putida strains for the control of crops diseases. This study will have implications in areas including, Ecology and Agriculture (root colonization), Medicine (antibacterial) and Basic Sciences.