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，每年在全世界造成数百万人死亡。更好地了解Tfp生物学是非常可取的，因为它将是开发新的预防和治疗方法的关键。Tfp介导细菌粘附于宿主细胞和表面，是宿主定殖的关键，但目前对Tfp介导粘附的分子机制还知之甚少，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，这些亚基可能在人类机会致病菌S的Tfp中的粘附中起直接作用。血，它会导致感染性心内膜炎，这是一种危及生命的疾病。在本项目中，我们将采用高通量基因组学、分子遗传学、蛋白质生物化学和结构生物学相结合的多学科方法，对这两种新发现的粘附素PilB和PilC进行详细的研究。总之，由于对少数公开的S.血链球菌基因组中PilB和PilC的序列变异性可能具有重要的功能性。将对血细胞进行测序，以确定这两种粘附素的变异程度，并观察是否与临床症状相关。然后，使用分子遗传学，这些序列变异的功能后果将在体内进行分析。最后，将代表性变体蛋白纯化，体外功能分析并提交结构分析。这有望使人们对Tfp介导的粘附有一个新的认识，并可能成为设计新药物干扰Tfp介导的重要人类细菌病原体定殖的第一步。

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.