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

嗜肺军团菌是一种革兰氏阴性细菌，会导致军团病，这是一种经常致命的肺炎。它在细胞外表面分泌一种独特的蛋白质--军团菌胶原样蛋白(LCL)。这既可以识别宿主细胞表面的硫酸糖胺聚糖(GAG)，介导黏附，也可以通过结合环境中的二价阳离子来促进生物膜的形成。LCL由一个负责在分泌后将其锚定在细菌表面的N-末端区域(LCL-NTR)、一个广泛的胶原样区域(它延伸到细菌表面之外)和一个独特的C-末端结构域(LCL-CTD)组成。我们已经确定了LCL-CTD的晶体结构，并用核磁共振和分子动力学模拟来了解它是如何与GAG(如肝素、软骨素)结合的。使用生化和生物物理技术，我们已经展示了镁是如何结合并引起变化的，这使得LCL-CTD能够识别脂多糖(LPS)。脂多糖是细菌膜的主要成分，这种结合使相邻的细菌聚集在一起。我们现在将使用核磁共振和冷冻-EM分别研究LCL-NTR+/-洗涤剂和LCL-CTD+/-内毒素的结构。然后，我们希望通过MD模拟了解它们在人工嗜肺乳杆菌膜中的行为，并使用生物膜分析利用嗜肺乳杆菌中LCL的靶点突变来验证这些发现。首席主管(加内特)将负责生物化学、结构生物学和细菌工作，副主管(洛伦茨)将领导项目的MD方面。这个PHD项目的具体目标是：AIM1：使用冷冻-EM来确定有/没有内毒素结合AIM2+/LCL-CTD的结构AIM2：通过核磁共振来阐明有/没有去污剂的LCL-NTR的结构Aim3：使用MD Aim4：检测这些复合体在人工膜中的行为使用MD Aim4：测试在嗜肺乳杆菌中产生的靶向LCL突变的理论

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