The T6SS toxins are powerful weapons for Pseudomonas' antibacterial strategy

细菌已经进化出许多策略来在恶劣的环境中茁壮成长，在敌对的宿主中定居，并在激烈的商品和生存竞争中与其他微生物竞争。感染人类的细菌病原体可以使用各种策略。它们会发展成急性感染，在短时间内致命，但也会建立慢性感染并在宿主体内持续一生。这是囊性纤维化（CF）患者肺部感染的情况。在感染的早期阶段，存在多种细菌，例如葡萄球菌、伯克霍尔德氏菌或铜绿假单胞菌。在患者生命的后期阶段，留下的唯一微生物是铜绿假单胞菌，其是慢性建立的并且将导致患者死亡。铜绿假单胞菌建立慢性感染的能力不仅限于CF或肺部疾病，而且还源于临床设置中使用的医疗器械的污染，并且铜绿假单胞菌在其上建立对抗生素治疗几乎不敏感的弹性生物膜。铜绿假单胞菌是一种成功的定植菌和致病微生物，其原因是多方面的。然而，最近的一项发现指出了它使用分子武器的能力，这有助于消除细菌竞争对手，如CF肺定植。这种武器是第六型分泌系统（T6 SS），其设计是一种刺穿目标生物体包膜的穿刺装置，以便注射致命毒素。为了保护自己免受T6 SS毒素的侵害，细菌产生解毒剂或免疫力，从而防止自我杀伤，并且仅针对缺乏这些免疫力的非自我生物体。T6 SS毒素的库目前被低估，并且由于以下几个原因，破译完整的T6 SS毒素库将是非常宝贵的。首先，使T6 SS成为强大武器的不是枪，而是它发射的毒素子弹。这些毒素的数量和不同程度将决定细菌定植的效力和效率。我们将使用完全随机化方法（TraDIS）鉴定所有T6 SS毒素/免疫对。利用这些知识，我们将能够确定任何临床分离株的T6 SS谱，从而了解和理解为什么特定分离株可能比另一种分离株更差。T6 SS景观的制图可能是感染背景和严重程度的指标。其次，鉴定功能未知的毒素可能会导致我们迫切需要的新药的开发，因为抗生素管道已经枯竭。换句话说，T6 SS毒素通过瞄准尚未探索的目标来杀死细菌，将为制药公司提供一个新的探索场所和药物设计的可能性。由于铜绿假单胞菌具有三种不同的T6 SS，每种都可能发射数十种不同的毒素，因此新发现的潜力是巨大的和令人兴奋的。总之，该项目不仅旨在解开铜绿假单胞菌可用的T6 SS毒素/免疫对的所有库，而且还旨在（i）了解这些毒素是否被所有三种T6 SS机器同时发射，（ii）可视化T6 SS如何停靠在猎物细胞上以及毒素如何穿过它，（iii）使用超分辨率荧光显微镜可视化毒素的运输，（iv）将这些知识应用于来自CF患者的临床分离株，以监测高T6 SS效力与宿主定殖和持久性的高能力之间的相关性。关于T6 SS机制的基本知识是至关重要的，因为人们可以预见为人类精心制作活的无害生物体的可能性，这些生物体可以配备能够消除细菌病原体的特定T6 SS武器，而这只有通过深入了解才有可能。

Bacteria have evolved plenty of strategies to thrive within harsh environments, to colonize hostile hosts and to compete with other microorganisms in a fierce race for goods and survival. Bacterial pathogens that infect humans can use various strategies. They develop acute infections, which turn fatal in a short period of time, but also establish chronic infections and persist within the host over a lifetime. That is the case for lung infections in cystic fibrosis (CF) patients. At early stages of infection, diverse bacteria are present, e.g. Staphylococcus, Burkholderia or Pseudomonas aeruginosa. At later stages in the patient life the sole microorganism left is P. aeruginosa, which is chronically established and will lead to patient death. The ability of P. aeruginosa to establish chronic infection is not exclusive to CF or pulmonary diseases but also results from contamination of medical devices used in clinical set up and on which P. aeruginosa establish a resilient biofilm barely sensitive to antibiotic treatments. From there it disseminates in the human body and causes high level of morbidity and mortality.There are multiple reasons for which P. aeruginosa is such a successful colonizer and pathogenic organism. However, one recent discovery pointed to its ability to use a molecular weapon, which facilitates the elimination of bacterial competitors such as in the CF lung colonization. This weapon is the type VI secretion system (T6SS) and has the design of a puncturing device perforating the envelope of target organisms in order to allow injection of lethal toxins. To protect itself from T6SS toxins the bacterium produces an antidote, or immunity, thus preventing self-killing and only aiming at non-self organisms lacking these immunities.The repertoire of T6SS toxins is currently undervalued and it will be invaluable to decipher the complete T6SS toxin armoury for several reasons. In the first place what makes the T6SS a potent weapon is not the gun but the toxin bullets it fires. How many and how distinct these toxins are will define how potent and efficient in colonization the bacterium is. We will use a completely randomized approach (TraDIS) to identify all T6SS toxin/immunity pairs. Using this knowledge we will be in a position to determine the T6SS profile of any clinical isolates and thus gain knowledge and understanding on why specific isolates may be worse than another. The cartography of the T6SS landscape could be an indicator of the context and severity of the infection. Secondly, the identification of toxins of unknown function could result in the development of new drugs that we desperately need considering the exhaustion of the antibiotic pipeline. In other words a T6SS toxin that kill bacteria by aiming at a yet unexplored target will provide pharmaceutical companies with a novel venue to explore and the possibility for drug design. Since P. aeruginosa possesses three distinct T6SSs, each potentially firing dozens of distinct toxins, the potential for new discoveries is huge and exciting.In summary, not only this project aims at unraveling the repertoire of T6SS toxin/immunity pairs available to P. aeruginosa, but it also aims (i) at understanding whether these toxins are fired at once and simultaneously by all three T6SS machines, (ii) at visualizing how the T6SS docks onto prey cells and how toxins travels through it, (iii) at visualising the transport of the toxins using super resolution fluorescence microscopy, (iv) at applying this knowledge to clinical isolates from CF patients in monitoring a correlation between high T6SS potency and high capability of host colonisation and persistence. Basic knowledge about the T6SS mechanism is crucial since one can foresee the possibility to elaborate live innocuous organisms for human kind, which could be equipped with a specific T6SS weaponry able to eliminate bacterial pathogens, and this will only be possible by having such in depth understanding.