Mislocalization of PBPs in Staphylococcus aureus gdpP mutant contributes to β-lactam resistance and surface protein cross-wall trafficking.

GdpP is a phosphodiesterase that degrades the second messenger c-di-AMP, which plays a key role in osmoregulation and antibiotic resistance in many gram-positive bacteria. Mutations in gdpP are frequently associated with mecA-independent β-lactam resistance in Staphylococcus aureus, but the mechanisms remain unknown. Here, we show that deletion of gdpP diminished cross-wall deposition of YSIRK+ surface protein A (SpA). Strikingly, all four native PBPs (PBP1-4) were mislocalized as distinct single foci in ΔgdpP cells. Deficiency in cell division initiation accumulated non-dividing cells in ΔgdpP, and the aberrant PBP foci were predominantly found at the cell periphery of non-dividing cells. The mislocalization of PBPs is due to the accumulation of c-di-AMP, as shown in both ΔgdpP and dacA (c-di-AMP synthase) overexpression strains. The mislocalized PBPs were largely inactive as they were unable to incorporate FDAAs locally. ΔgdpP did not show significant change in overall peptidoglycan cross-linking. Consistently, ΔgdpP strains were resistant to β-lactams that target PBPs, but not to vancomycin that targets peptidoglycan cross-linking. Finally, the aberrant PBP foci formation correlates with resistance to β-lactams-mediated killing, but not to vancomycin. Based on these results, we propose that increased c-di-AMP level in ΔgdpP cells leads to aggregation of PBPs into peripheral foci that mis-target β-lactams and delay in cell division dysregulates YSIRK+ protein cross-wall deposition. Our results provide new mechanistic insight into connections among c-di-AMP signaling, β-lactam resistance, and YSIRK+ virulence protein assembly that are widely found in gram-positive bacteria.IMPORTANCEC-di-AMP signaling pathway is widely found in bacteria and frequently associated with antibiotic resistance and tolerance. In Staphylococcus aureus, mutations in gdpP encoding the phosphodiesterase that degrades c-di-AMP have been associated with mecA-independent β-lactam resistance, while the mechanisms are largely unknown. Here, we report new findings that accumulation of c-di-AMP in the gdpP mutant leads to aberrant foci formation of all four native PBPs in S. aureus, which correlates with resistance to β-lactam antibiotics. Moreover, the gdpP mutant is deficient in cell division, which diminishes cross-wall assembly of YSIRK+ surface virulence proteins. Our study provides new insight into connections among c-di-AMP signaling, β-lactam resistance, and YSIRK+ virulence protein biogenesis that are widely found in gram-positive bacteria, and lays a foundation for developing new therapeutics.