Biochemical and Structural Characterization of LiaR from Vancomycin-Resistant E. faecalis: the ‘Master Regulator’ of the Cell-Envelope Stress Response

By: Milya G. Davlieva1, Yiwen Shi1, Michael Zianni2, Troy Johnson3, Paul Leonard3, John Ladbury3, Cesar A. Arias4,5 and Yousif Shamoo1.
1Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005 USA; Plant-Microbe Genomics Facility, The Ohio State University, Columbus, OH, USA; 3Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 4Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX, USA; 5Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, TX, USA. Introduction CESR: Cell Envelope Stress Response through liaFSR signaling Conclusions

Introduction

The CDC estimates there are over 66,000 enterococcus infections per year in the United States (CDC, 2013) and has classified vancomycin resistant enterococci (VRE) as a serious threat. In the absence of a good alternative, the cyclic lipopeptide daptomycin (DAP) has been used “off-label” for the treatment of multidrug-resistant enterococci infections and has become a key “front-line” antimicrobial against these organisms. However, the emergence of daptomycin resistance during therapy has become increasingly frequent and will increase as DAP is used more broadly. Previously, has been shown that DAP resistance in enterococci is linked to mutations in genes that alter the cell envelope stress response (CESR) (liaFSR)1. Mutations to genes encoding the proteins comprising the liaFSR two-component signaling pathway occur early in adaptation and are a critical first step toward DAP resistance. LiaR is a ‘response regulator’ in the cell envelope stress response pathway that regulates several downstream operons essential to adaptation. Among the most critical downstream regulatory targets of LiaR is LiaX (formally called yvlb)2. In this study, we report the expression, purification, and biophysical characterization of LiaR and an adaptive variant LiaRD191N from E. faecalis that is associated with DAP resistance. Altogether, we have elucidated a mechanistic basis for how mutations in LiaR confer increased resistance to DAP though alterations in signaling.

CESR: Cell Envelope Stress Response through liaFSR signaling

cell-envelope-stress-response

  • In the absence of cell-membrane acting antibiotics, liaFSR system is turned “OFF”; LiaF inhibits LiaS mediated signaling
  • LiaS responds to membrane stress by phosphorylation of LiaR
  • Activation of CESR system via LiaR results in a cascade of events that allow bacteria to adapt to the antibiotic attack and survive (i.e., develop clinical resistance)
  • LiaR proteins and homologues are putative response regulators that harbor two domains: N-terminal signal receiver domain and Cterminus DNA-binding motif

E. faecalis S613 LiaR and its variants were successfully over-expressed Using using EnPresso™ Cultivation Set

s613-liar

  • LiaRD50E -conserved active-site Asp 50 was replaced with a Glu in order to produce the constitutively active mutant
  • LiaRD50A - nonphosphorylatable variant in order to be able to capture the response regulator LiaR in an inactive conformation
  • LiaRD191N - an adaptive mutant that confers increased daptomycin resistance in E. faecalis S613

Daptomycin mutant LiaRD191N mimics some of the properties that would be expected of LiaR in an activated (phosphorylated) state

liar-phosphorylated

  • A comparison of the DNA sequences upstream of the liaFSR and liaXYZ operons and comparable operons from B. subtilis and S. aureus suggested that LiaR has an T(X)4C(X)4G(X)4A consensus motif
  • Relative affinity of LiaR for the liaFSR promoter compared with the liaXYZ promoter appears to be weaker
  • A putative phosphomimetic mutant of LiaR showed increased affinity for DNA targets suggesting that phosphorylation state is a critical component of LiaR specificity
  • Mutation of the putative phosphorylation site to alanine (LiaRD50A) decreased LiaR affinity for either sequence below detectable levels
  • The adaptive mutant LiaRD191N enhanced binding of LiaR to the liaFSR and liaXYZ operons

The D191N mutation in LiaR promotes higher order complex formation

d191n-mutation

Sedimentation equilibrium analytical ultracentrifugation profiles for each protein depicted by the black lines. The residuals for each fit are provided in the lower panel, below the experimental data.

Overall structure of DBD LiaRD191N bound to the consensus sequence within the liaXYZ operon

overall-structure-of-dbd

  • In order to elucidate the structural basis for LiaR affinity for specific DNA targets, we have solved the high resolution structure of the DNA binding domain LiaRD191N complexed to double-stranded DNA by X-ray at 2.4 Å.
  • Crystal structures of the LiaR DNA binding domain bound to the consensus sequence site suggest that LiaR binding significantly bends DNA

Conclusions

  • Mutation of the putative site of phosphorylation Asp-50 to Glu strongly favors formation of a tetramer with high affinity for the consensus DNA recognition sequences within liaXYZ operon
  • The adaptive mutation LiaRD191N shifts LiaR into the activated tetramer even in the absence of phosphorylation leading to a constitutively “on” state
  • As a master regulator of the cell envelopes stress response LiaR, as well as components of the LiaFRS system, may prove to be excellent targets for the development of new strategies and drugs
  • Altogether, we have elucidated a mechanistic basis for how mutations in LiaR confer increased resistance to DAP though alterations in signaling

Materials

     

References

  1. Arias, C.A., Panesso, D., McGrath, D.M., Qin, X., Mojica, M.F., Miller, C., Diaz, L., Tran, T.T., Rincon, S., Barbu, E.M., et al. (2011). Genetic basis for in vivo daptomycin resistance in enterococci. N Engl J Med 365, 892-900.
  2. Miller, C., Kong, J., Tran, T.T., Arias, C.A., Saxer, G., and Shamoo, Y. (2013). Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance. Antimicrob Agents Chemother 57, 5373-5383.

 

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