The bacterial defensin resistance protein MprF consists of separable domains for lipid lysinylation and antimicrobial peptide repulsion.
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.
Ernst
Christoph M
CM
Cellular and Molecular Microbiology Division, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany.
Staubitz
Petra
P
Mishra
Nagendra N
NN
Yang
Soo-Jin
SJ
Hornig
Gabriele
G
Kalbacher
Hubert
H
Bayer
Arnold S
AS
Kraus
Dirk
D
Peschel
Andreas
A
D019881:Aminoacyltransferases / Q000737:chemistry* / Q000502:physiology; D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides / Q000037:antagonists & inhibitors*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D017576:Daptomycin / Q000494:pharmacology; D057131:Immune Evasion; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins; D010715:Phosphatidylglycerols / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013211:Staphylococcus aureus / Q000472:pathogenicity*
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D008565:Membrane Proteins; D010715:Phosphatidylglycerols; D019881:Aminoacyltransferases; C429146:mprF protein, Staphylococcus aureus; D008239:Lysine; D017576:Daptomycin