Understanding DNA-binding by type IV pilins: key event during transformation in naturally competent bacteria

自然转化是一种生物学过程，在此过程中，细菌从周围环境中捕获游离DNA，并将其整合到基因组中，通过获得新基因进行遗传改变。这一过程对人类健康具有重要意义，因为它促进了细菌之间的基因水平转移，当它导致细菌病原体出现时，可能会产生破坏性的医学后果，这些病原体具有更高的毒力和/或对抗生素的抗性。自然转化的第一步是DNA摄取，由称为Tff的表面细丝介导，Tff由称为IV型菌毛蛋白的亚基组成。这些丝状体非常重要，因为它们（i）在原核生物中几乎是普遍存在的，（ii）与一系列非常不同的特性有关，其中许多特性是细菌毒力的关键，这些细菌会引起重要的人类疾病，如霍乱，脑膜炎，淋病，在这个抗生素耐药性不断上升的时代，研究Tff生物学具有极大的医学意义，因为这可能最终导致新的抗病毒药物干扰Tff组装或Tff介导的功能。直到最近，转化的最早步骤，Tff如何与DNA相互作用还知之甚少，因为还没有鉴定出Tff相关的DNA受体。我们最近的工作提供了一个突破，显示IV型菌毛蛋白相关的脑膜炎球菌ComP显示内在的DNA结合能力，并结合DNA在一个新的方式。这为理解IV型菌毛蛋白在自然转化过程中如何结合DNA铺平了道路，这是本研究提案的首要目标。我们将结合遗传学，生物化学和结构学方法，并在此方面拥有丰富的经验，我们将实现以下三个目标。目标1：在原子水平上确定ComP菌毛蛋白如何结合DNA以及不同的直系同源物如何能够区分密切相关的DNA基序。目的2：目的3：鉴定感受态（假）皮利中的DNA结合菌毛蛋白，该菌毛蛋白是天然转化过程中分布最广的Tff。

Natural transformation is the biological process during which bacteria capture free DNA from their surroundings and incorporate it into their genomes to become genetically altered by acquiring new genes. This process has important implications for human health because it promotes horizontal transfer of genes between bacteria, which can have devastating medical consequences when it leads to the emergence of bacterial pathogens with increased virulence and/or resistance to antibiotics.The first step during natural transformation, DNA uptake, is mediated by surface filaments named Tff composed of subunits called type IV pilins. These filaments are extremely important because they (i) are almost universal in prokaryotes, and (ii) have been associated with a fascinating array of very different properties, many of which are key for the virulence of bacteria causing important human diseases such as cholera, meningitis, gonorrhoea, diarrhea etc. Studying Tff biology is of great medical interest in this era of rising antibiotic resistance since this could ultimately lead to the development of new antivirulence drugs interfering with Tff assembly or Tff-mediated functions.Until recently, the earliest step in transformation, how Tff interact with DNA was poorly understood because no Tff-associated DNA receptor had ever been identified. Our recent work provided a breakthrough by showing that type IV pilins related to meningococcal ComP display intrinsic DNA-binding ability and bind DNA in a novel fashion. This paves the way to an atomic level understanding of how type IV pilins bind DNA during natural transformation, which is the overarching objective of this research proposal.Using a combination of genetic, biochemical, and structural approaches for which we have extensive experience, we will address the following three aims.Aim 1: determine at an atomic level how ComP pilins bind DNA and how different orthologs are able to distinguish between closely related DNA motifs.Aim 2: characterise the recently identified Pasteurellaceae DNA-binding pilin, unrelated to ComP, to determine how it binds DNA.Aim 3: identify the DNA-binding pilin in competence (pseudo)pili, the most widespread Tff involved in natural transformation.