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

View ORCID ProfilePei Yi Choo, Charles Y. Wang, View ORCID ProfileMichael S. VanNieuwenhze, View ORCID ProfileKimberly A. Kline
doi: https://doi.org/10.1101/2021.07.23.451969
Pei Yi Choo
aSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
bSchool of Biological Sciences, Nanyang Technological University, Singapore, Singapore
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Charles Y. Wang
aSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
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Michael S. VanNieuwenhze
cDepartments of and Chemistry, Indiana University, Bloomington, Indiana, USA
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Kimberly A. Kline
aSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
bSchool of Biological Sciences, Nanyang Technological University, Singapore, Singapore
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  • For correspondence: kkline@ntu.edu.sg
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Abstract

Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One virulence factor is the sortase-assembled endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that the pilus sortase covalently links pilus monomers prior to recognition, while the housekeeping sortase cleaves at the LPXTG motif within the terminal pilin subunit, and subsequently attaches assembled pilus fiber to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) throughout the E. faecalis cell cycle via immunofluorescence microscopy and fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of the E. faecalis diplococcus were completely saturated with Ebp, while Ebp appeared as two foci directly adjacent to the nascent septum in the newer hemisphere. A similar localization pattern was observed for another cell wall anchored substrate by sortase A, aggregation substance (AS), suggesting that this may be a general rule for all SrtA substrates in E. faecalis. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that binds and sequesters lipid II cell wall precursors, new Ebp deposition at the cell surface was not disrupted. These data suggest an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto un-crosslinked cell wall, independent of new PG synthesis.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 24, 2021.
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Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis
Pei Yi Choo, Charles Y. Wang, Michael S. VanNieuwenhze, Kimberly A. Kline
bioRxiv 2021.07.23.451969; doi: https://doi.org/10.1101/2021.07.23.451969
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Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis
Pei Yi Choo, Charles Y. Wang, Michael S. VanNieuwenhze, Kimberly A. Kline
bioRxiv 2021.07.23.451969; doi: https://doi.org/10.1101/2021.07.23.451969

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