The bacterial defensin resistance protein MprF consists of separable domains for lipid lysinylation and antimicrobial peptide repulsion

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.

Figure 1. Structure of MprF and truncated proteins.

A) Predicted trans-membrane topology of S. aureus MprF with amino acid positions predicted to form TMSs indicated. B) Truncated MprF proteins used to study the function of MprF. Length and calculated molecular weight of MprF variants are shown. Construction of plasmids is described in detail in Table S1.

Figure 2. TLC and Western Blot analysis of E. coli expressing truncated or mutated variants of MprF.

A) Polar lipids from strains containing expression plasmids without insert (control), with full-length mprF, or truncated mprF genes encoding proteins without the indicated TMS were separated by TLC and stained with the aminogroup-specific dye ninhydrin. B) The cytosolic fraction of E. coli strains expressing MprF(−14) and the membrane fractions of strains expressing MprF(−12), MprF(−10), MprF(−8) or containing the empty expression plasmid pET28 (control) were subjected to immunoblot analysis with a His-tag-specific antibody. The TMSs-containing proteins migrated slightly faster than expected, which is probably due to increased SDS binding capacity and/or incomplete unfolding of TMSs . Molecular weight standard proteins are shown at the right margin. C) TLC analysis of E. coli strains expressing MprF variants with alanine exchanges. Polar lipids from strains containing the expression plasmid pET28a without insert (control), with unaltered mprF(−8), or with variants encoding proteins with the indicated amino acid exchanges were separated by TLC and stained with the aminogroup-specific dye ninhydrin. Positions of phosphatidylethanolamine (PE) and lysylphosphatidylglycerol (Lys-PG) are indicated in A) and C).

Figure 3. Impact on Lys-PG production and resistance to antimicrobial peptides of MprF variants in S. aureus ΔmprF.

A) Lys-PG content in S. aureus wild-type (WT) or ΔmprF strains from logarithmic (log) or stationary growth phase containing the indicated plasmids were separated by TLC, stained with the phosphate groups-specific dye molybdenum blue, and quantified densitometrically. B) Minimal inhibitory concentrations (MICs) of CAMPs such as α-defensins HNP1-3, cathelicidin LL-37, gallidermin, and daptomycin. Means and SEM of three (HNP1-3, gallidermin, daptomycin) or two (LL-37) independent experiments are shown. MICs of HNP 1-3 and LL-37 for WT and ΔmprF with plasmid pRB474mprF were above the highest tested concentration of 300 µg/ml. Therefore, significances could only be calculated for gallidermin and daptomycin. C) Impact on Lys-PG content and daptomycin susceptibility of ΔmprF containing different expression vectors for the Lys-PG synthase domain MprF(−8). *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant versus WT (A), ΔmprF containing plasmid pRB474mprF (B), or ΔmprF containing plasmid pRB474mprF(−8) (C).

Figure 4. Impact on Lys-PG production and resistance to antimicrobial peptides of MprF(−8) and MprF(−C) expressed in trans.

The two protein domains were expressed on separate plasmids [pRB474mprF(−8) and pTX15mprF(−C)] in S. aureus ΔmprF. A) Polar lipids were separated by TLC and stained with the aminogroup-specific dye ninhydrin. B) Minimal inhibitory concentrations of gallidermin and daptomycin. pRB474 and pTX16 are empty control plasmids. Means and SEM of three independent experiments are shown. ***, P<0.001; ns, not significantly different versus S. aureus ΔmprF containing plasmid pRB474mprF and pTX16.

Figure 5. Impact of the hydrophobic N-terminal domain of MprF on the ability of Lys-PG to repulse cationic cytochrome c and to reach the outer leaflet of the cytoplasmic membrane.

A) The capacities of S. aureus wild-type (WT) and ΔmprF (left panel) or ΔmprF containing the indicated plasmids (right panel) to bind cytochrome c were compared. B) Inner and outer-leaflet localization of Lys-PG in ΔmprF bearing the indicated plasmids was determined by analyzing the ability of the membrane-impermeable fluorescent dye fluorescamine to react with Lys-PG. pRB474 and pTX16 are empty control plasmids. Means and SEM of three (A) and four to eight replicas from two (B) independent experiments are shown. *, P<0.05; **, P<0.01; ns, not significantly different versus S. aureus WT (A, left panel) or ΔmprF containing plasmids pRB474mprF and pTX16 (A, right panel).

Figure 6. Model for the mode of MprF-mediated bacterial CAMP resistance.

Lys-PG is synthesized from Lys-tRNA and PG by the synthase domain of MprF. Lys-PG can only neutralize the outer surface of the membrane upon translocation to the outer cytoplasmic membrane leaflet, which is facilitated by the large N-terminal integral membrane domain of MprF.