Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis

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.

Monoderm bacteria: the new frontier for type IV pilus biology

“…Prepilins (major and minor) are first translocated via the SRP‐Sec pathway across the cytoplasmic membrane (CM) (Arts et al , ; Francetic et al , ), where they remain initially as bitopic proteins with their α1N behaving as a transmembrane domain. Their leader peptide is then processed after the conserved Gly by the prepilin peptidase (Aly et al , ), which also methylates the first residue of mature pilins as confirmed in S. sanguinis (Berry et al , ). It is unclear at this stage, if the minor pilins are assembled first and cap the filaments as proposed for other Tff (Korotkov and Hol, ).…”

“…B) and 3D structure (Fig. C) (Berry et al , ). It remains to be seen whether such a heteropolymeric structure might be a more general property of Tfp in monoderms, which awaits purification and biochemical characterisation of filaments in other species.…”

“…S. sanguinis Tfp, which have a Tfp morphology (Fig. A), were purified to homogeneity and shown to be heteropolymers of two similar pilins (PilE1 and PilE2) in a 4:3 ratio (Berry et al , ). These pilins exhibit canonical class III signal peptide (Fig.…”

“…As in diderms, filament retraction is powered by the retraction of ATPase PilT, which is dispensable for piliation (Gurung et al , ). It is important to underline that the term ‘minor pilin’ is just generic, only indicating that the corresponding proteins are cleaved by the prepilin peptidase to become low abundance components of the filaments (Berry et al , ). This does not imply that these proteins play identical or even similar functions in each system, which is supported by the fact that minor pilins in different systems share no sequence homology.…”

“…It is not known which pilus component mediates binding, but the presence of very large (>50 kDa) minor pilins with a ‘modular’ architecture, that is, with bulky C‐terminal functional domains, is certainly an intriguing possibility. In S. sanguinis , PilB and PilC contain C‐terminal domains often involved in adhesion in proteins unrelated to Tfp, a von Willebrand factor type A domain (IPR002035) and a concanavalin A‐like lectin/glucanase structural domain (SSF49899), respectively (Berry et al , ). This suggests that S. sanguinis Tfp harbour two minor pilins with intrinsic adhesive properties, maybe at their tip, allowing the bacterium to bind host protein and carbohydrate ligands.…”