In depth biochemical and structural analysis of a Gram-positive type IV pilus

Type IV pili (Tfp), which belong to a large class of filamentous nanomachines called type IV filaments (Tff), are surface-exposed and functionally versatile filaments, widespread in prokaryotes. Although Tfp have been extensively studied in several Gram-negative pathogens where they are key virulence factors, many aspects of their biology remain poorly understood. Here, we have performed an in depth biochemical and structural analysis of Tfp in the opportunistic pathogen Streptococcus sanguinis , which has recently emerged as a Gram-positive model for the study of these filaments. In particular, we have focused on the five pilins and pilin-like proteins involved in Tfp biology in this species (PilA, PilB, PilC, PilE1 and PilE2). We found that the two major pilins (PilE1 and PilE2) (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments, (ii) display only one of the post-translational modifications frequently found in type IV pilins, i.e. a processed and methylated N-terminus, (iii) are found in the same hetero-polymeric filaments, and (iv) are not functionally equivalent with respect to twitching motility. The 3D structure of PilE1, which we solved by nuclear magnetic resonance (NMR), reveals a classical pilin fold with an uncommon highly flexible C-terminus. Intriguingly, PilE1, which fits well into available Gram-negative Tfp structures, is more structurally similar to pseudopilins forming short Tff than bona fide Tfp-forming major pilins, further underlining the evolutionary relatedness between different Tff. Finally, we show that S. sanguinis Tfp also contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB and PilC. By providing the first global biochemical and structural picture of a Gram-positive Tfp, our findings have important implications for widespread filamentous nanomachines.