Journal of Molecular Biology
Volume 432, Issue 20, 18 September 2020, Pages 5681-5695
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Enterococcal PrgA Extends Far Outside the Cell and Provides Surface Exclusion to Protect against Unwanted Conjugation

https://doi.org/10.1016/j.jmb.2020.08.018Get rights and content
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Highlights

  • Conjugative plasmids often provide mechanisms to block uptake of the same plasmid.

  • PrgA from pCF10 decreases cellular adhesion and provides surface exclusion.

  • PrgA presents a protease domain 40 nm outside the cell wall.

  • PrgA contributes to cell fitness.

  • With these results, we explain the molecular function of PrgA.

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.

Keywords

crystallography
conjugation
cellular adhesion
protease
T4SS

Abbreviations

MGE
mobile genetic element
T4SS
type 4 secretion system
ITC
isothermal titration calorimetry

Cited by (0)

A.S. and H.H. shared the first authorship position.