Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

Enterococcus faecalis virulence requires cell wall-associated proteins, including the sortase-assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co-localize with newly synthesized PG. Instead, surface-exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.

Automated summary

Enterococcus faecalis relies on cell wall-associated proteins for virulence, specifically relying on Ebp for biofilm formation. The traditional understanding is that sortases attach substrates to lipid II peptidoglycan precursors before they are incorporated into the growing cell wall. However, this study finds that surface-exposed Ebp in E. faecalis does not co-localize with newly synthesized peptidoglycan. Instead, Ebp is localized to the older cell hemisphere and excluded from sites of new peptidoglycan synthesis. This challenges the current paradigm and proposes a new model where sortase substrate deposition can occur on older peptidoglycan, independent of new cell wall synthesis.