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Mutations in\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      are frequently associated with\n                      <jats:italic toggle=\"yes\">mecA<\/jats:italic>\n                      -independent \u03b2-lactam resistance in\n                      <jats:italic toggle=\"yes\">Staphylococcus aureus<\/jats:italic>\n                      , but the mechanisms remain unknown. Here, we show that deletion of\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      diminished cross-wall deposition of YSIRK+ surface protein A (SpA). Strikingly, all four native PBPs (PBP1\u20134) were mislocalized as distinct single foci in \u0394\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      cells. Deficiency in cell division initiation accumulated non-dividing cells in \u0394\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      , 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 \u0394\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">dacA<\/jats:italic>\n                      (c-di-AMP synthase) overexpression strains. The mislocalized PBPs were largely inactive as they were unable to incorporate FDAAs locally. \u0394\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      did not show significant change in overall peptidoglycan cross-linking. Consistently, \u0394\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      strains were resistant to \u03b2-lactams that target PBPs, but not to vancomycin that targets peptidoglycan cross-linking. Finally, the aberrant PBP foci formation correlates with resistance to \u03b2-lactams-mediated killing, but not to vancomycin. Based on these results, we propose that increased c-di-AMP level in \u0394\n                      <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                      cells leads to aggregation of PBPs into peripheral foci that mis-target \u03b2-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, \u03b2-lactam resistance, and YSIRK+ virulence protein assembly that are widely found in gram-positive bacteria.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        C-di-AMP signaling pathway is widely found in bacteria and frequently associated with antibiotic resistance and tolerance. In\n                        <jats:italic toggle=\"yes\">Staphylococcus aureus<\/jats:italic>\n                        , mutations in\n                        <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                        encoding the phosphodiesterase that degrades c-di-AMP have been associated with\n                        <jats:italic toggle=\"yes\">mecA<\/jats:italic>\n                        -independent \u03b2-lactam resistance, while the mechanisms are largely unknown. Here, we report new findings that accumulation of c-di-AMP in the\n                        <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                        mutant leads to aberrant foci formation of all four native PBPs in\n                        <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                        , which correlates with resistance to \u03b2-lactam antibiotics. Moreover, the\n                        <jats:italic toggle=\"yes\">gdpP<\/jats:italic>\n                        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, \u03b2-lactam resistance, and YSIRK+ virulence protein biogenesis that are widely found in gram-positive bacteria, and lays a foundation for developing new therapeutics.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01430-26","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T13:00:39Z","timestamp":1786539639000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Mislocalization of PBPs in\n                    <i>Staphylococcus aureus gdpP<\/i>\n                    mutant contributes to \u03b2-lactam resistance and surface protein cross-wall 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