The Bacterial Defensin Resistance Protein MprF Consists of Separable Domains for Lipid Lysinylation and Antimicrobial Peptide Repulsion

PLoS Pathog. 2009 Nov;5(11):e1000660. doi: 10.1371/journal.ppat.1000660. Epub 2009 Nov 13.

Abstract

Many bacterial pathogens achieve resistance to defensin-like cationic antimicrobial peptides (CAMPs) by the multiple peptide resistance factor (MprF) protein. MprF plays a crucial role in Staphylococcus aureus virulence and it is involved in resistance to the CAMP-like antibiotic daptomycin. MprF is a large membrane protein that modifies the anionic phospholipid phosphatidylglycerol with l-lysine, thereby diminishing the bacterial affinity for CAMPs. Its widespread occurrence recommends MprF as a target for novel antimicrobials, although the mode of action of MprF has remained incompletely understood. We demonstrate that the hydrophilic C-terminal domain and six of the fourteen proposed trans-membrane segments of MprF are sufficient for full-level lysyl-phosphatidylglycerol (Lys-PG) production and that several conserved amino acid positions in MprF are indispensable for Lys-PG production. Notably, Lys-PG production did not lead to efficient CAMP resistance and most of the Lys-PG remained in the inner leaflet of the cytoplasmic membrane when the large N-terminal hydrophobic domain of MprF was absent, indicating a crucial role of this protein part. The N-terminal domain alone did not confer CAMP resistance or repulsion of the cationic test protein cytochrome c. However, when the N-terminal domain was coexpressed with the Lys-PG synthase domain either in one protein or as two separate proteins, full-level CAMP resistance was achieved. Moreover, only coexpression of the two domains led to efficient Lys-PG translocation to the outer leaflet of the membrane and to full-level cytochrome c repulsion, indicating that the N-terminal domain facilitates the flipping of Lys-PG. Thus, MprF represents a new class of lipid-biosynthetic enzymes with two separable functional domains that synthesize Lys-PG and facilitate Lys-PG translocation. Our study unravels crucial details on the molecular basis of an important bacterial immune evasion mechanism and it may help to employ MprF as a target for new anti-virulence drugs.

Publication types

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

MeSH terms

  • Aminoacyltransferases / chemistry*
  • Aminoacyltransferases / physiology
  • Anti-Bacterial Agents
  • Antimicrobial Cationic Peptides / antagonists & inhibitors*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / physiology
  • Daptomycin / pharmacology
  • Immune Evasion*
  • Lysine / chemistry
  • Membrane Proteins
  • Phosphatidylglycerols / metabolism*
  • Protein Structure, Tertiary
  • Protein Transport
  • Staphylococcus aureus / pathogenicity*

Substances

  • Anti-Bacterial Agents
  • Antimicrobial Cationic Peptides
  • Bacterial Proteins
  • Membrane Proteins
  • Phosphatidylglycerols
  • Aminoacyltransferases
  • mprF protein, Staphylococcus aureus
  • Lysine
  • Daptomycin