Structural studies into biofilm formation by the Legionella pneumophila collagen-like protein

嗜肺军团菌胶原蛋白样生物膜形成的结构研究

• 批准号: 2723191
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2723191
• 关键词: Structural; studies; into; biofilm; formation

Legionella pneumophila is a Gram-negative bacterium that causes Legionnaires'disease, an often-fatal pneumonia. It secretes a unique protein onto its extracellularsurface; the Legionella collagen-like (Lcl) protein. This functions in both recognisingsulphated glycosaminoglycans (GAGs) on the host cell surface which mediatesadhesion, but also promotes biofilm formation through binding environmental divalentcations. Lcl is formed of an N-terminal region (Lcl-NTR) responsible for anchoring it tothe bacterial surface after its secretion, an extensive collagen-like region whichextends it away from the surface, and a unique C-terminal domain (Lcl-CTD). We havedetermined the crystal structure of Lcl-CTD and have used NMR and MD simulations tounderstand how it binds GAGs (e.g. heparin, chondroitin). Using biochemical andbiophysical techniques we have shown how magnesium binds and causes changeswhich enables Lcl-CTD to recognise lipopolysaccharides (LPS). LPS is a majorcomponent of bacterial membranes, and this binding allows adjacent bacteria to thenaggregate. We will now use NMR and cryo-EM to study the structures of the Lcl-NTR+/- detergent and Lcl-CTD +/- LPS, respectively. We then wish to understand how theybehave within an artificial L. pneumophila membrane using MD simulations andvalidate these findings using targeted point mutations of Lcl in L. pneumophila usingbiofilm assays. The primary supervisor (Garnett) will be responsible for biochemistry,structural biology and bacterial work, the secondary supervisor (Lorenz) will lead theMD aspects of the project. The specific aims of this PhD project are to: Aim1: Use cryo-EM to determine the structure of Mg2+/Lcl-CTD with/without LPS bound Aim2:Elucidate the structure of Lcl-NTR with/without detergent by NMR Aim3: Monitor howthese complexes behave within an artificial membrane using MD Aim4: Test theoriesthat arise with targeted lcl mutations in L. pneumophila

嗜肺军团菌是一种革兰氏阴性细菌，可引起军团菌病，这是一种经常致命的肺炎。它分泌一种独特的蛋白质到其细胞外表面;军团菌胶原样（Lcl）蛋白。它的功能是识别宿主细胞表面的硫酸化糖胺聚糖（GAG），介导粘附，还通过结合环境二价阳离子促进生物膜形成。Lcl由N-末端区（Lcl-NTR）、胶原样区（Lcl-CTD）和C-末端区（Lcl-CTD）组成，N-末端区负责在分泌后将Lcl锚定到细菌表面，胶原样区将Lcl延伸远离表面。我们已经确定了Lcl-CTD的晶体结构，并使用NMR和MD模拟来了解它如何结合GAG（例如肝素，软骨素）。使用生物化学和生物物理技术，我们已经显示了镁如何结合并引起变化，使Lcl-CTD能够识别脂多糖（LPS）。LPS是细菌膜的主要成分，这种结合使相邻的细菌聚集在一起。我们现在将分别使用NMR和cryo-EM来研究Lcl-NTR+/-去污剂和Lcl-CTD +/- LPS的结构。然后，我们希望了解它们在人工L中的行为。pneumophila膜的分子生物学模拟，并验证了这些发现使用靶点突变的Lcl在L。pneumophila使用生物膜测定。主要主管（Garnett）将负责生物化学、结构生物学和细菌工作，次要主管（Lorenz）将领导项目的MD方面。本博士项目的具体目标是：Aim 1：使用cryo-EM来确定Mg 2 +/Lcl-CTD的结构，有/没有LPS结合Aim 2：通过NMR阐明Lcl-NTR的结构，有/没有去污剂Aim 3：监测这些复合物如何在人工膜内使用MD Aim 4：测试理论，在L.嗜肺