Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation and virulence

  1. Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden
  2. Wallenberg Centre for Molecular Medicine, Umeå University, Umeå, Sweden

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.

Read more about eLife’s peer review process.


  • Reviewing Editor
    Michael Eisen
    University of California, Berkeley, Berkeley, United States of America
  • Senior Editor
    Michael Eisen
    University of California, Berkeley, Berkeley, United States of America

Reviewer #1 (Public Review):

Sun and colleagues outline structural and mechanistic studies of the bacterial adhesin PrgB, an atypical microbial cell surface-anchored polypeptide that binds DNA. The manuscript includes a crystal structure of the Ig-like domains of PrgB, cryo-EM structures of the majority of the intact polypeptide in DNA-bound and free forms, and an assessment of the phenotypes of E. faecalis strains expressing various PrgB mutants.

Generally, the study has been conducted with a good level of rigor, and there is consistency in the findings. However, I do have some specific technical concerns relating to the study. Although the PX work has been expertly undertaken, the Cryo-EM structures reported are both at ~10-angstrom resolution. Visual inspection indicates that the positioning of the PrgB domains into the EM envelopes is somewhat questionable and this needs to be addressed. The narrative of the manuscript very much hinges on this being correct. In addition, wrt the PrgB mutant studies, it could be that the loss of function observed in specific mutants is simply a consequence of mutation-induced misfolding of those polypeptides. Experimental evidence supporting the direct interaction between the PAD and the stalk domains in PrgB is also lacking.

Reviewer #2 (Public Review):

Having previously solved the X-ray crystallographic structure of the polymer adhesin domain (PAD) of PrgB from E. faecalis, the authors looked to build on that work by crystallizing a nearly full-length construct of PrgB. Though they were successful in their crystallization endeavors, the crystal contained only what was previously thought to be two domains with RGD motifs. The authors' high-resolution structure shows that in fact the C-terminal portion of PrgB is made up of four immunoglobulin-like domains. The authors then set out to collect single-particle cryoEM data in a bid to obtain a full-length structure of PrgB, both in the presence and absence of ssDNA. The authors were only able to obtain quite low-resolution data, which they fit their crystal structures into. The authors then used these structures to inform the design of novel deletion mutants and point mutations, as well as to rationalize years of phenotypic data from other published mutants.

The X-ray crystallographic structure is beautiful and in combination with their in vivo data allowed them to propose a model where PrgB positions cells at an appropriate distance for conjugation. The cryoEM data are not convincing in their current state, and I, therefore, don't believe that their model of the immunoglobulin domains acting to protect the PAD domain of PrgB from PrgA is well supported. Perhaps there are 2D classes or other data that make a case for the fit of the crystal structures into the cryoEM volumes, but without a PAD deletion or perhaps a dataset including a PAD-specific antibody, I don't feel the fit is supported.

The in vivo experiments appear to be done well and the authors' discovery that the Ser-Asn-Glu is not important for generalized aggregation but has an additional yet unknown role in conjugation and biofilm formation is exciting and well supported by their data.

Author Response:

First of all, we would like to thank the reviewers for their work. We appreciate the constructive review comments and useful suggestions to further improve our article.

The main criticism on our manuscript, from both reviewers, is that the cryo-EM structures are of low resolution and that the fit of the crystallographic structures of the PAD and the stalk domain into these low-resolution structures is questionable. We would like to point out that the cryo-EM data, and the conclusions from it, are not essential for the main conclusions of the article. All mutants that we made in this study were designed based on the structural data obtained from the high-resolution X-ray structures, with no input from the low-resolution cryo-EM docked models. We chose to include the cryo-EM data since it allowed us to speculate about the interaction between the PAD and the stalk domain of PrgB, domains that we have separately determined the structures of via X-ray crystallography. We agree with the reviewers that further experiments are needed to verify this potential interaction. Therefore, we will perform additional biochemical assays to investigate the proposed interaction. We will also try to optimize the cryo-EM data to hopefully allow for a more reliable fit of our high-resolution crystallographic structures. Once that is done, we will submit a revised version of the manuscript.

On behalf of all authors,

Ronnie Berntsson

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation