Functional analysis of the proteins and multiprotein complexes involved in the biogenesis of type IV pili

微生物是地球上数量最多的生物体。它们可以在包括人类在内的其他生物体中引起疾病，但也可以在日常人类活动中有所帮助，比如消化。一些用于执行特定的工业或制造过程，如生产药物或合成激素，而另一些用于有机废物的生物转化。所有这些特性的一个共同特征是微生物必须与其他有机体或其自然和人造环境相互作用。为此，它们通常使用被称为菌毛的毛状表面细胞器，其中IV型菌毛(TFP)是最普遍的。然而，TFP是如何组装的，以及它们如何调节它们所连接的多种功能(与各种表面的黏附、聚集、运动和DNA导入)仍然知之甚少。因此，我们试图用脑膜炎奈瑟氏菌作为模式细菌来回答这些问题，脑膜炎奈瑟氏菌是一种重要的人类病原体，导致全球50%的细菌性脑膜炎病例。除了帮助开发针对人类病原体的更有效的治疗方法并更好地了解一个基本的生物学问题外，我们的工作还可能为其他不太容易处理的医学或商业上重要的表达TFP的物种带来潜在的应用和好处。在第一步中，我们已经确定了TFP组装中涉及的所有基因，这表明相应的机制是复杂的，因为它由15个不同的蛋白质组成。这表明，只有通过简化论的方法才有可能理解这台机器的工作原理，即首先检查它的较小部分，这可以在重新组装后解释整个过程。因此，我们使用细菌遗传学来确定功能性TFP的表达需要四个不同的步骤，并且我们可以定义上述15种蛋白质中的每一种都参与其中的哪一步。这项研究项目的目标是多方面的，目的是了解蛋白质如何在上述四个步骤中作为亚基一起工作，以组装功能性TFP。首先，我们将确定这15种蛋白质的基本生化性质，如teir丰度和在细胞中的定位。然后，我们将确定相互作用的蛋白质，以便将拼图的碎片重新组合在一起。这将使用两种不同和互补的技术来完成。最后，我们将进入下游阶段，即确定单个蛋白质组分的三维结构和功能模式。我们将从研究PilW蛋白开始，它具有特别有趣的特性。

Microbes are the most numerous living organism on the planet. They can cause diseases in other organisms, including humans, but they can also be helpful in everyday human activities, like digestion. Some are used to perform specific industrial or manufacturing processes such as the production of drugs or synthetic hormones, while others are used for the bioconversion of organic waste. A common trait for all these propertie is the necessity for microbes to interact with other organisms or their natural and man-made environments. For this purpose, they often use hair-like surface organelles known as pili, among which type IV pili (Tfp) are the most widespread. However, how Tfp are assembled and how they mediate the multiple functions they are linked with (adhesion to various surfaces, aggregation, motility and DNA import) remains poorly understood. We are therefore trying to answer these questions using as a model bacterium Neisseria meningitidis, which an important human pathogen responsible for 50% of the cases of bacterial meningitis worldwide. Besides helping develop more efficient therapies against human pathogens and leading to a better understanding of a fundamental biological problem, our work may lead to potential applications and benefits for other less tractable medically or commercially important species that express Tfp. In a first step, we have identified all the genes involved in Tfp assembly, which indicated that the corresponding machinery is complex since it numbers fifteen different protein components. This suggested that understanding how this machinery works would only be possibe through a reductionist approach, that is by first examining smaller pieces of it, which could explain the whole upon re-assembly. Therefore, we used bacterial genetics to determine that four different steps are necessary for the expression of functional Tfp and we could define at which one of these steps each of the above fifteen proteins is involved. The objectives of this research project are multiple and aim at understanding how the protein work together as subgroups in the above four steps to assemble functional Tfp. As a first thing, we will determine the basic biochemical properties of these fifteen proteins, such as teir abundance and localization in the cell. We will then identify the proteins that are interacting with each other in order to put the pieces of the puzzle back together. This will be done using two different and complementary techniques. Finally, we will move to a downstream stage which consists in determining the tri-dimensional structure and mode of function of the individual protein components. We will start by studying the PilW protein, which has particularly interesting properties.