Enterococcal PrgA Extends Far Outside the Cell and Provides Surface Exclusion to Protect against Unwanted Conjugation

Andreas Schmitt, Helmut Hirt, Michael A. Järvå, Wei Sheng Sun, Josy ter Beek, Gary M. Dunny, Ronnie P.A. Berntsson

Research output: Contribution to journalArticlepeer-review

Abstract

Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via type 4 secretion systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a second copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggests that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

Original languageEnglish (US)
Pages (from-to)5681-5695
Number of pages15
JournalJournal of Molecular Biology
Volume432
Issue number20
DOIs
StatePublished - Sep 18 2020

Bibliographical note

Funding Information:
The authors thank Dr. Eric Geertsma for providing the plasmids of the FXCloning system and Prof. Peter J Christie for valuable discussions regarding the project. We are grateful to the beamline scientists at the European Synchrotron Radiation Facility for providing assistance in using beamline ID29 and ID30-A. This work was supported by grants from the Swedish Research Council ( 2016-03599 ), Knut and Alice Wallenberg Foundation and Kempestiftelserna ( JCK-1524 & SMK-1869 ) to R.P-A.B., and by PHS grant R35 GM118079 from the National Institutes of Health to G.M.D.

Publisher Copyright:
© 2020 The Authors

Keywords

  • T4SS
  • cellular adhesion
  • conjugation
  • crystallography
  • protease

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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