Deciphering type IV pilus biology in the Gram-positive opportunistic pathogen Streptococcus sanguinis

Abstract

Type IV pili (Tfp) are the paradigm of a large group of diverse and functionally versatile nanomachines, intensively studied in Gram-negative bacteria. However, details regarding the molecular mechanisms of Tfp biogenesis and/or mediated functions are still unclear. Thus, owing to the inherent lack of outer cell wall in Gram-positive bacteria, my PhD has focused on molecular characterisation of Tfp in a simpler such bacterium Streptococcus sanguinis. My work has shown that the naturally competent S. sanguinis produces bona fide retractable Tfp enabling twitching motility, but dispensable for competence. Unlike Gram-negative Tfp, we show that S. sanguinis Tfp are unusual since they are composed of two pilin proteins, a feature likely to be shared by other Gram-positive Tfp-expressing species. All the genes involved in Tfp biology in S. sanguinis are found within a pil locus encoding 21 proteins. A systematic genetic study highlighted that 10 proteins only are required for Tfp biogenesis, whilst another four modulate twitching motility. To enhance genetic manipulation of S. sanguinis, a markerless mutagenesis strategy was devised enabling us to make various mutations in situ, which helped us characterise some of these proteins further. Via this methodology, the last six genes of the pil locus were found to be completely dispensable for Tfp biology. To get an overall structural picture of Tfp in S. sanguinis, the structure of one of the major pilins (PilE1) was determined by NMR. Moreover, three pilin-like proteins within the pil locus were found to be minor Tfp components. Collectively, my work has established S. sanguinis as a robust Gram-positive model organism for studying Tfp, which paves way for interesting future studies.