Results 
The graRS mutant shows altered cell surface charge but unaltered lysyl-phosphatidylglycerol (LPG) content
In order to study if reduced expression of the dlt operon upon graRS disruption results in altered cell surface charge, we compared binding of the red-coloured, cationic protein cytochrome c to wild type S. aureus SA113 (WT), the isogenic graRS mutant, and the plasmid-complemented mutant.
The graRS mutant bound significantly more cytochrome c than the WT or the complemented mutant (Fig. 1A), which is in accordance with the recently described reduced content of D-alanine residues in the teichoic acids of the mutant [10].
To analyse whether increased binding of cytochrome c by the graRS mutant is in fact due to altered cell surface charge by decreased teichoic acid alanylation or the altered expression of other surface-exposed molecules, we also examined binding of cytochrome c to a dltA deletion mutant, which completely lacks D-alanine substitution of teichoic acids [7], and a dltA/graRS double deletion mutant, which was generated by transducing the graRS mutation into the dltA mutant.
Due to the high binding capacities of the dltA and the dltA/graRS mutant we modified the conditions in order to prevent limitation of applied cytochrome c.
Deletion of the graRS genes in the dlt mutant background did not lead to further increased binding of cytochrome c compared to the dltA mutant (Fig. 1B).
This finding indicates that the increased binding of cytochrome c by the graRS mutant is due to decreased alanylation of teichoic acids and that the resulting surface charge alteration does not result from other regulatory effects mediated by GraRS.
Increased binding of cationic proteins may also result from reduced mprF expression and, accordingly, reduced lysylphosphatidylglycerol (LPG) content.
To control for this possibility, we compared patterns of membrane lipids from log-phase bacteria by thin-layer chromatography.
The amounts of LPG from WT and graRS mutant were indistinguishable (data not shown), which corroborates recent findings that mprF is not among the graRS-regulated genes in S. aureus SA113 [10].
The graRS mutant is more susceptible to killing by LL-37 and human neutrophil granulocytes in vitro In an attempt to test whether the increased affinity of the graRS mutant to cationic molecules leads to higher susceptibility to human host defense peptides, we compared inactivation of WT and graRS mutant by the human cathelicidin LL-37.
This antimicrobial peptide is active against S. aureus at high concentrations or long exposure [15] but in this experiment we chose conditions under which the LL-37 did not affect viability of the S. aureus WT.
Accordingly, the WT and complemented mutant strains showed no significant decrease in CFU following LL-37 exposure, whereas the number of graRS mutant bacteria recovered was only 25% of the original inoculum (Fig. 2A).
Next we investigated whether the graRS mutant is killed faster than the parental strain by human neutrophils, which produce high amounts of LL-37 and other CAMPs as components of their antibacterial killing arsenal.
The graRS mutant was killed by neutrophils considerably faster than the WT strain.
After 15 and 30 min, the recovered CFU of the graRS mutant were significantly lower than those of the WT (Fig 2B).
Of note, our previous studies had shown that altered alanylation of teichoic acids does not affect the rate of PMN phagocytosis [9].
Taken together, these data indicate that graRS-mediated control of CAMP resistance mechanisms is of importance for S. aureus evasion from neutrophil killing.
