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.
Highlights
In order to combat increasingly antibiotic-resistant bacteria such as Staphylococcus aureus, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and enterococci new antimicrobial strategies based on compounds with anti-virulence or anti-fitness properties are increasingly in the focus of research efforts [1,2]
Certain bacterial immune-evasion factors such as the multiple peptide resistance factor (MprF) protein are highly conserved in many bacterial pathogens and represent attractive targets for new ‘anti-virulence’ drugs
MprF modifies bacterial membrane lipids with the amino acid L-lysine, which leads to electrostatic repulsion of the membrane-damaging peptides
Summary
In order to combat increasingly antibiotic-resistant bacteria such as Staphylococcus aureus, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and enterococci new antimicrobial strategies based on compounds with anti-virulence or anti-fitness properties are increasingly in the focus of research efforts [1,2]. Bacterial immune evasion mechanisms such as the mprF or dltABCDencoded pathways are conserved over a wide range of bacterial species thereby representing attractive targets for broadly active antimicrobial compounds that would not kill the bacteria directly but render them susceptible to endogenous host defense molecules [3,4]. The occurrence of closely related immune evasion factors in many bacterial pathogens is reflected by the conserved nature of the most critical antimicrobial host defense molecules. The MprF and DltABCD proteins protect many bacterial pathogens against CAMPs by reducing the negative net charge of bacterial cell envelopes [3,8]. The dltABCD operon products neutralize polyanionic teichoic acid polymers by esterification with D-alanine in many Gram-positive bacteria [9]
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