The T6SS as a search engine for naturally validated antibacterial targets

细菌对所有临床相关抗生素都出现了耐药性。尽管人们普遍认为这是一场全球危机，但迄今为止实施的大多数策略都令人失望地不成功，特别是那些基于合理药物设计和目标识别方法的策略。一个基本和关键的问题确实是定义什么是好的抗菌靶标，以便它能够有效地适用于药物设计。数十亿年来，微生物已经进化出最好的方式来击败其他人，以获得空间和营养。他们已经确定了细菌的寄生虫，并设计了相应的策略来抑制相应的分子途径，这些分子途径将立即挑战细菌的生长和生存。这就是人们可以认为是自然验证的药物靶点。细菌VI型分泌系统（T6 SS）是通过毒素注射杀死竞争对手的终极武器。大多数特征性的T6 SS毒素作用于细胞壁、膜、核酸，这些都是我们目前大多数抗生素的经典靶标。我们刚刚开始了解这个系统是如何工作的，T6 SS毒素只是冰山一角。同样重要的是要认识到，产生T6 SS毒素的细菌也会产生特异性免疫蛋白，特异性地保护免受每种T6 SS毒素的活性。一个关键的观察结果是，编码毒素的基因和编码免疫力的基因在细菌基因组中被发现为彼此相邻的串联基因对。该项目旨在开发被广泛忽视的天然抗菌剂库，也就是说，细菌已经进化出大量的T6 SS毒素，以有效地杀死细菌的竞争对手，我们相信这些毒素存在的数量要大得多，并且具有意想不到的广泛范围，多样的生物化学活动。虽然T6 SS毒素本身不太可能在短期内成为有效的治疗药物，但它们为我们指出的天然有效的抗菌靶点将具有很大的价值。一种使用T6 SS竞争并杀死敌人的强效革兰氏阴性细菌是铜绿假单胞菌，这是一种在WHO病原体名单上排名靠前的微生物，对抗菌药物耐药性（AMR）至关重要。在这里，我们将使用铜绿假单胞菌作为模型来进行T6 SS毒素的系统搜索，这些毒素使用标准基因组和生物信息学分析无法识别。在以前的工作中，我的实验室实施了一个遗传筛选（TraDIS），以确定转座子突变敏感的T6 SS。这些突变体在不同的免疫基因中受到影响，并且相邻基因的表征将导致鉴定新的T6 SS毒素。我们的筛选得到了充分验证，因为它允许鉴定已知的毒素/免疫对，并且证明在鉴定未怀疑的T6 SS毒素及其免疫方面非常有效。我们在本发明中提出充分表征的T6 SS毒素之一被称为Tse 8，并且被怀疑干扰蛋白质合成，特别是通过阻碍转酰胺体复合物的功能，导致Asn-和Gln-的缺乏，发现新的毒素并确定它们的作用机制将为制药公司提供一个可用的抗菌靶点金矿，未来此外，了解细菌如何以我们可能不会怀疑的方式相互战斗是准备翻译影响所需要的。

Bacterial resistance has emerged to all clinically relevant antibiotics. Despite the widespread recognition of this as a global crisis the majority of the strategies implemented thus far have been disappointingly unsuccessful, notably those based on rational drug design and target identification approaches. One fundamental and key issue is indeed to define what a good antimicrobial target is so that it would effectively be amenable to drug design. Over billions of years microbes have evolved the best ways to out-compete others to gain access to space and nutrients. They have identified bacterial Achille's heels and designed strategies to inactivate corresponding molecular pathways that would right away challenge bacterial growth and survival. This is what one can consider as naturally validated drug targets.The bacterial Type VI Secretion System (T6SS) is the ultimate weapon killing competitors by toxins injection. Most characterized T6SS toxins work against cell wall, membrane, nucleic acids, which are all classic targets for most of our current antibiotics. We are at the beginning of understanding how this system works and the characterized T6SS toxins represent only the tip of the iceberg. It is also important to realise that the bacteria that produces T6SS toxins would also produce specific immunity proteins that specifically protect against the activity of each individual T6SS toxin. One key observation is that the gene encoding the toxin and the gene encoding the immunity are found on the bacterial genome as tandem gene pairs next to each other.This project aims at exploiting the largely overlooked reservoir of natural antibacterials, namely the plethora of T6SS toxins that bacteria have evolved to effectively kill bacterial competitors and which we believe exist in a much larger number and have an unexpected broad range of diverse biochemical activity. While T6SS toxins themselves are unlikely to be effective therapeutics in the short term, the naturally validated antibacterial targets they point us towards would be of great value.A potent gram-negative bacterium using the T6SS to outcompete and kill foes is Pseudomonas aeruginosa, an organism which is high on the WHO list of pathogens that are critical for Anti-Microbial Resistance (AMR). Here, we will use P. aeruginosa as model to perform a systematic search for T6SS toxins, which are not identifiable using standard genomic and bioinformatic analysis. In previous work my laboratory implemented a genetic screen (TraDIS) to identify transposon mutants sensitive to the T6SS. These mutants are affected in distinct immunity genes and the characterization of the adjacent gene would lead to the identification of a novel T6SS toxin. Our screen was fully validated since it allowed to identify the toxin/immunity pairs which were already known and proved to be very effective at the identification of unsuspected T6SS toxins and their immunities. One of the T6SS toxin which we propose to fully characterize in the present proposal is called Tse8 and is suspected to interfere with protein synthesis, notably by hampering function of the transamidosome complex which leads to a shortage in Asn- and Gln-tRNA.Finding new toxins and determining their mechanisms of action is going to offer a gold mine of usable antibacterial targets that pharmaceutical companies would be able to consider in the future. Furthermore, understanding how bacteria fight each other in ways we might not suspect is what is needed to prepare translational impact.