Abstract
Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis. 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 pilins, i.e. a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.
Highlights
Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff)
Bioinformatic analysis of the proteins encoded by the pil locus in S. sanguinis, which contains all the genes involved in Tfp biology (24), predicts five pilins and/or pilinlike proteins (PilA, PilB, PilC, PilE1, and PilE2) (Fig. S1A)
Visual inspection of the sequences of the remaining proteins suggests that PilA has a degenerate class III signal peptide (Fig. 1B), which is not identified by the bioinformatic tools available, including PilFind, which is dedicated to the identification of type IV pilins (27)
Summary
Type IV pili; Tff, type IV filament(s); PTM, post-translational modification(s); T2SS, type II secretion system; TH, Todd Hewitt; LB, lysogeny broth; TEM, transmission electron microscopy; cryoEM, cryo-electron microscopy; sPCR, splicing PCR; AGC, automated gain control; HCD, higher-energy collisional induced dissociation; NCE, normalized collision energy; aa, amino acid(s). Tfp are highly dynamic filaments, constantly extending and retracting. Retraction has been best characterized in a subclass known as Tfpa, where it results from filament depolymerization powered by the cytoplasmic hexameric ATPase PilT (18), which generates massive tensile forces (19, 20). A comprehensive genetic analysis of S. sanguinis Tfpa (24) has revealed that they: (i) are assembled by a similar machinery as in Gram-negative species but with fewer components, (ii) are retracted by a PilT-dependent mechanism, generating tensile forces very similar to those measured in Gram-negative species, and (iii) power intense twitching motility. We have focused on the pilins and pilin-like proteins involved in Tfp biology in S. sanguinis and have performed a global biochemical and structural analysis of its filaments
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