Table 1.
Characteristics of the daptomycin-exposed clinical strains of Staphylococcus aureus used in this study.
Table 2.
Characteristics of the daptomycin-exposed laboratory strains of Staphylococcus aureus used in this study.
Figure 1.
Percentage of daptomycin-exposed pairs (n = 12) with a mutation in each functional gene category.
All daptomycin-nonsusceptible isolates had at least one mutation in a gene coding for phospholipid biosynthesis, including mprF, cls2 or pgsA.
Table 3.
Predicted protein changes in clinical- and laboratory-derived daptomycin-nonsusceptible isolates of Staphylococcus aureus.
Figure 2.
Phospholipid biosynthesis genes are integral to the development of reduced susceptibility to daptomycin in S. aureus.
(A) The predicted amino acid changes associated with the 11 SNPs identified within mprF. (B) The five mutations identified in cls2 were mapped to four positions in the protein, all within the two N-terminal transmembrane domains. (C) Six mutations were identified in pgsA and were mapped to four positions in the protein, three of which were in transmembrane domains. The ‘A’ numbers correspond to the daptomycin-nonsusceptible isolates, and the arrows point to the position of the amino acid change.
Figure 3.
Schematic of our working hypothesis for the functional effect of the observed mutations.
mprF mutations lead to an increase in lysinylation of phosphotidylglycerol (PG) to form L-PG, and an increase in translocation of this positively charged L-PG to the outer leaflet of the membrane, leading to electrorepulsion of daptomycin. In isolation, or in concert with mprF mutations, mutations in cls2 may lead to altered membrane charge or effect binding of daptomycin to the membrane. Finally, PgsA is important in the initial step of phospholipid biosynthesis, converting CDP-diacylglycerol (CDP-DG) into PG.
Figure 4.
Transmission electron microscopy of two clinical pairs showing a thickening of the cell wall in the daptomycin-nonsusceptible isolates.
(A) and (B) represent A8819 (daptomycin-susceptible) and A8817 (daptomycin-nonsusceptible), respectively. (C) and (D) represent A8796 (daptomycin-susceptible) and A8799 (daptomycin-nonsusceptible), respectively. P<0.001 for both.