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

自然转化是细菌从周围环境中捕获游离DNA并将其整合到自己的基因组中，通过获得新基因进行基因改变的生物过程。这一过程对人类健康具有重要影响，因为它促进了细菌之间的基因水平转移，当它导致毒力和/或抗药性增强的细菌病原体出现时，可能会产生毁灭性的医学后果。自然转化的第一步，DNA摄取，是由名为TFF的表面细丝介导的，该表面细丝由称为IV型菌毛的亚基组成。这些细丝非常重要，因为(I)它们几乎在原核生物中普遍存在，(Ii)它们与一系列迷人的非常不同的性质有关，其中许多性质是引起霍乱、脑膜炎、淋病、腹泻等重要人类疾病的细菌毒力的关键。在这个抗生素耐药性不断上升的时代，研究TFF生物学具有重大的医学意义，因为这最终可能导致新的抗毒药物的开发，干扰Tff组装或Tff介导的功能。直到最近，Tff如何与DNA相互作用仍知之甚少，因为尚未发现与Tff相关的DNA受体。我们最近的工作取得了突破，表明与脑膜炎双球菌CoMP相关的IV型菌毛蛋白显示出内在的DNA结合能力，并以一种新的方式结合DNA。这为原子水平上理解IV型菌毛蛋白在自然转化过程中如何与DNA结合铺平了道路，这是本研究计划的首要目标。我们将使用我们拥有丰富经验的遗传、生化和结构方法的组合，解决以下三个目标。目标1：在原子水平上确定CoMP菌毛蛋白如何与DNA结合，以及不同的同源物如何区分密切相关的DNA基序。目标2：表征最近发现的与CoMP无关的巴氏杆菌DNA结合菌毛蛋白，以确定它如何与DNA结合。目标3：鉴定DNA结合毛细蛋白能力(伪)菌毛蛋白，这是自然转化中最广泛的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.