Competence pilus biogenesis in Streptococcus pneumoniae

Sammanfattning: Horizontal gene transfer drives bacterial evolution and is crucial for the pathogenicity of many bacteria that can exchange genetic material through distinct mechanisms such as natural transformation. S. pneumoniae, a common asymptomatic colonizer of the human nasopharynx, is naturally transformable, being able to take up naked DNA and integrate it into its genome. Natural transformation allows pneumococci to acquire new traits, which can provide fitness advantages and contribute to the modulation of carriage and invasive diseases such as pneumonia, meningitis or septicaemia. Natural transformation is a complex and highly regulated process, which requires bacteria to enter in a distinct metabolic state named competence. During this period, competent pneumococci express all proteins required for transformation. Among them, are the proteins encoded by the comG operon (comGA-G) implicated in the assembly of pneumococcal competence type IV pili (T4P), which were shown to directly bind DNA. Deciphering how these filaments are assembled is a crucial step in understanding pneumococcal natural transformation and the main focus of this thesis. Pneumococcal T4P can be several micrometres long and are composed of the major pilin ComGC. We demonstrated that ComGC has intrinsic capacity to polymerize and to assemble into pili. We also solved the high-resolution NMR structure of N-terminal truncated ComGC and found that ComGC has distinct structural features compared to other known type IV pilins and is highly flexible. Based on the structure of ComGC, we further identified a region with potential DNA binding properties, providing the basis to understand the function of competence pili. In addition to our investigation of the major pilin ComGC, we also studied the role of the minor pilins ComGD, ComGE, CopmGF and ComGG in pilus assembly and transformation. We showed that the pneumococcal minor pilins form a minor pilin complex likely to prime pilus assembly. Moreover, we visualized that ComGF is part of the filament and present strong evidence for the role of ComGG as the linker between the minor pilin complex and ComGC. Pneumococcal colonization and virulence can be influenced by different factors including induction of competence genes and cell morphology. We found that Deterministic Lateral Displacement is a suitable label-free microfluidics technique to sort S. pneumoniae into subpopulations based on morphological properties, which can be a relevant tool to understand the mechanisms governing colonization and how bacteria cause disease. In summary, here we provide a genetic and molecular characterization of the key components involved in pneumococcal pilus assembly and present a novel technique to study S. pneumoniae subpopulations.