MprF-mediated biosynthesis of lysylphosphatidylglycerol, an important determinant in staphylococcal defensin resistance

Abstract

Frequently bacteria are exposed to membrane-damaging cationic antimicrobial molecules (CAMs) produced by the host's immune system (defensins, cathelicidins) or by competing microorganisms (bacteriocins). Staphylococcus aureus achieves CAM resistance by modifying anionic phosphatidylglycerol with positively charged L-lysine, resulting in repulsion of the peptides. Inactivation of the novel S. aureus gene, mprF, which is found in many bacterial pathogens, has resulted in the loss of lysylphosphatidylglycerol (L-PG), increased inactivation by CAM-containing neutrophils, and attenuated virulence. We demonstrate here that expression of mprF is sufficient to confer L-PG production in Escherichia coli, which indicates that MprF represents the L-PG synthase. L-PG biosynthesis was studied in vitro and found to be dependent on phosphatidylglycerol and lysyl-tRNA, two putative substrate molecules. Further addition of cadaverin, a competitive inhibitor of the lysyl-tRNA synthetases, or of RNase A abolished L-PG biosynthesis, thereby confirming the involvement of lysyl-tRNA. This study forms the basis for further detailed analyses of L-PG biosynthesis and its role in bacterial infections.

Low pH Enhances the Action of Maximin H5 against Staphylococcus aureus and Helps Mediate Lysylated Phosphatidylglycerol-Induced Resistance

“…It has recently been shown that increasing levels of Lys-PG and other lysylated phospholipids promote the rigidification and stabilisation of bacterial membranes through the reduction of electrostatic repulsion between anionic lipid head-groups, thereby inhibiting the ability of AMPs to penetrate these membranes 35,36 . Moreover, it is generally accepted that a contribution to the protective mechanism provided by Lys-PG derives from a 'charge effect' whereby the positively charged head-group carried by the lipid is able to reduce the net negative charge of host bacterial membranes thus inhibiting the binding of cationic AMPs to these membranes 27,28,31,[33][34][35][36][37][38] . It therefore seems likely that the contribution made by 'charge effects' to this protective mechanism is further enhanced by low pH given that the Lys-PG head-group is predominantly cationic under acid conditions but becomes more zwitterionic in character as pH is increased 29,93 .…”

“…According to this mechanism of resistance, cationic AMPs activate a sensor histidine kinase (ApsS) of the ApsSR (GraSR) regulon, which leads to the up-regulated expression of the virulence factor mprF. The protein product of this gene, mprF, is a Lys-PG synthase that catalyses the transfer of lysine from lysyl-tRNA to the PG head-group and flipping of the resulting Lys-PG to the outer leaflet of the S. aureus CM 23,[31][32][33][34] . The presence of the lipid in the CM of S.…”

“…aureus attenuates a number of membrane associated properties that inhibit the ability of cationic AMPs to bind and exert their membranolytic action against these membranes 27,28,31,[33][34][35][36][37][38] . This effect in enhanced by low pH conditions, when the production of Lys-PG is upregulated and where levels of the Lys-PG as high as 80 % of total membrane lipid have been reported for the organism 29,[39][40][41][42] .…”

The action mechanism of daptomycin

“…The reaction is catalysed by the membrane protein MprF, which also supplies the translocase activity that subsequently transfers lysyl-PG from the inner to the outer membrane leaflet. 92 The abundance of lysyl-PG in bacterial membranes correlates with resistance to both cationic antimicrobial peptides 93 and daptomycin. Gainof-function mutations in the mprF gene cause increased lysyl-PG levels, decreased PG levels, and daptomycin resistance in Staphylococcus aureus (see reference 94 and citations therein).…”

Monitoring Lys-tRNALys phosphatidylglycerol transferase activity

“…While the physiological significance of the MprF pathway has recently been explored, and qualitative in vitro TLC assays were set up using radiolabeled Lys (11), there is to date no convenient in vitro assay to quantitatively monitor the Lys-tRNA Lys PG transferase activity. Here we report general methods to express and obtain an enriched cellular fraction containing active MprF.…”