High-throughput comparative and functional genomic analysis of type IV pilus (Tfp) localised adhesins in the opportunistic human pathogen Streptococcc

TFP是许多人类细菌病原体的关键毒力因子，每年在全球范围内导致数百万人死亡。更好地了解TFP生物学是非常可取的，因为这将是开发新的预防和治疗方法的关键。TFP是宿主定植的关键，因为它们介导细菌与宿主细胞和表面的附着。不幸的是，TFP介导的黏附的分子机制尚不清楚，这阻碍了新治疗方法的设计。这个项目源于我们最近发现的亚基，这些亚基可能在机会性人类病原体血链球菌的TFP黏附中发挥直接作用，这种病原体会导致感染性心内膜炎，危及生命。在这个项目中，我们将使用最先进的多学科方法结合高通量基因组学、分子遗传学、蛋白质生物化学和结构生物学来详细表征这两个新发现的粘附素，命名为PilB和Pilc。总之，由于对几个公开可用的血链球菌基因组的初步比较基因组分析揭示了PilB和PilC中的一些序列变异，这可能具有重要的功能后果，因此将对大量的临床分离株进行测序，以确定这两种粘附素的变异程度，并看看是否与临床症状有关。然后，利用分子遗传学，这些序列变异的功能后果将在体内进行分析。最后，对具有代表性的变异蛋白进行纯化、体外功能分析和结构分析。这有望对TFP介导的黏附有一个先例的了解，并可能是设计干扰TFP介导的在重要人类细菌病原体中定植的新药的第一步。

Tfp are key virulence factors in many human bacterial pathogens thta cause millions ofdeaths each year worldwide. A better understanding of Tfp biology is highly desirable as itwill be crucial for developing new prevention and therapy methods. Tfp are key for hostcolonisation as they mediate attachment fo bacteria to host cells and surfaces.Unfortunately, the molecular mechanisms of Tfp-mediated adhesion are poorlyunderstood, which hinders the design of new treatments. This project stems from on ourrecent discovery of subunits that are likely to play a direct role in adhesion in the Tfp ofthe opportunistic human pathogen S. sanguinis, which causes infective endocarditis a lifethreateningcondition. During this project, we will use a state-of-the-art multi-disciplinaryapproach combining high-throughput genomics, molecular genetics, protein biochemistryand structural biology to characterise in detail these two newly discovered adhesins namedPilB and PilC. In brief, since a preliminary comparative genomic analysis on the fewpublicly available S. sanguinis genomes revealed some sequence variability in PilB andPilC, which might have important functional consequences, a large collection of clinicalisolates of S. sanguinis will be sequenced to determine the degree of variability of thesetwo adhesins and see if there is a correlation with clinical symptoms. Then, usingmolecular genetics, the functional consequences of these sequence variations will beanalysed in vivo. Finally, representative variant proteins will be purified, analysedfunctionally in vitro and submitted to structural analysis. This is expected to give anuprecedented understanding of Tfp-mediated adhesion and could be the first step towardsthe design of new drugs interfering with Tfp-mediated colonisation in important humanbacterial pathogens.