Abstract
Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.
Highlights
In bacteria, the adaptation to environmental conditions and sensing of the metabolic status of the cell is often mediated by two-component regulatory systems (TCS)
To test whether WalR abundance was impacted in the antisense strains, the antisense plasmids were transformed into S. aureus NRS384 walR-FLAG and a Western blot of the cell lysates using an anti-FLAG antibody was performed which showed no difference in WalR abundance (Figure 2a)
Because YycH abundance was reduced upon induction of the yycI antisense RNA and vice versa, we present only results of the yycHI double knockdown in comparison to the empty pEPSA5 vector control in the other experiments
Summary
The adaptation to environmental conditions and sensing of the metabolic status of the cell is often mediated by two-component regulatory systems (TCS). WalRK, is essential to maintain cell wall metabolism, mainly by controlling the transcription of cell wall lysing enzymes that are essential for growth [2,3,4]. First discovered in Bacillus subtilis [5] and later in S. aureus [6], this TCS comprises the membrane-bound histidine kinase WalK and the cytoplasmic response regulator WalR. The genes for two additional membrane proteins, yycH and yycI, are co-transcribed with walR and walK [7]. An additional cytoplasmic protein encoded by yycJ, a gene located downstream of the walRK operon is controlled by a separate promoter and does not show any functional relation to the WalRK TCS in S. aureus [7]
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