{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T01:10:33Z","timestamp":1768525833683,"version":"3.49.0"},"posted":{"date-parts":[[2025,6,29]]},"group-title":"Microbiology","reference-count":83,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,6,29]],"date-time":"2025-06-29T00:00:00Z","timestamp":1751155200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"National Institute of Health","award":["AI153521"],"award-info":[{"award-number":["AI153521"]}]},{"name":"National Institute of Health","award":["AI164794"],"award-info":[{"award-number":["AI164794"]}]},{"name":"National Institute of Health","award":["GM135095"],"award-info":[{"award-number":["GM135095"]}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Disease","doi-asserted-by":"crossref","award":["T32AI007001"],"award-info":[{"award-number":["T32AI007001"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  encodes a mutated variant of penicillin-binding protein 1 (PBP1) and is a key resistance determinant that increases the penicillin MIC (MIC\n                  <jats:sub>PEN<\/jats:sub>\n                  ) above the clinical breakpoint in\n                  <jats:italic>Neisseria gonorrhoeae<\/jats:italic>\n                  . Despite the removal of penicillin from treatment guidelines for gonococcal infections in the 1980s,\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  is present in nearly 50% of current\n                  <jats:italic>N. gonorrhoeae<\/jats:italic>\n                  isolates in the PubMLST database. Bioinformatic analysis indicates that\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  is exclusive to\n                  <jats:italic>N. gonorrhoeae<\/jats:italic>\n                  isolates, whereas Leu-421 is 100% conserved in other\n                  <jats:italic>Neisseria<\/jats:italic>\n                  species. To understand the involvement of\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  in antibiotic resistance, we introduced\n                  <jats:italic>ponA<\/jats:italic>\n                  variants encoding 16 different amino acids at position-421 into FA6140, a penicillin-resistant gonococcal isolate that naturally harbors\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  . Proline-421 was the only mutation that increased the MIC\n                  <jats:sub>PEN<\/jats:sub>\n                  to the same level as FA6140. We also assessed the fitness of strains with the 16 mutant\n                  <jats:italic>ponA<\/jats:italic>\n                  alleles over multiple serial passages, both with and without sub-MIC levels of penicillin. There was no fitness defect attributed to\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  under these experimental conditions; instead, our analyses suggest that the widespread occurrence of\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  is driven by its capacity to increase the MIC\n                  <jats:sub>pen<\/jats:sub>\n                  above the clinical breakpoint. In FA6140 transformed with the mosaic\n                  <jats:italic>penA<\/jats:italic>\n                  allele from strain H041, a ceftriaxone-resistant isolate,\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  increased the MIC of ceftriaxone, suggesting that ceftriaxone targets PBP1 in this strain. We conclude that the\n                  <jats:italic>ponA<\/jats:italic>\n                  <jats:sup>L421P<\/jats:sup>\n                  allele emerged in gonococcal isolates, increasing the MIC\n                  <jats:sub>PEN<\/jats:sub>\n                  above the clinical breakpoint, and has remained in the population even after the removal of penicillin from treatment guidelines.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    The emergence of antibiotic-resistant\n                    <jats:italic>Neisseria gonorrhoeae<\/jats:italic>\n                    threatens effective treatment of gonorrhea, one of the most common sexually transmitted infections worldwide. Understanding the genetic changes that drive and maintain resistance is crucial for anticipating future resistance trends. Here, we investigated the impact of a key resistance mutation in PBP1 (encoded by\n                    <jats:italic>ponA<\/jats:italic>\n                    <jats:sup>L421P<\/jats:sup>\n                    ). Although penicillin has not been used to treat gonorrhea for decades, this mutation remains widespread even in recent\n                    <jats:italic>N. gonorrhoeae<\/jats:italic>\n                    isolates.\n                    <jats:italic>ponA<\/jats:italic>\n                    <jats:sup>L421P<\/jats:sup>\n                    confers clinically relevant penicillin resistance without imposing an\n                    <jats:italic>in vitro<\/jats:italic>\n                    fitness cost.\n                    <jats:italic>ponA<\/jats:italic>\n                    <jats:sup>L421P<\/jats:sup>\n                    also increases resistance to ceftriaxone in strains with\n                    <jats:italic>penA<\/jats:italic>\n                    alleles that are associated with ceftriaxone resistance. This work highlights the role of the\n                    <jats:italic>ponA<\/jats:italic>\n                    <jats:sup>L421P<\/jats:sup>\n                    allele in shaping the current antibiotic resistance landscape and supports the need for ongoing surveillance and evolutionary studies of such mutations in the gonococcal population.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.06.27.662027","type":"posted-content","created":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T00:00:14Z","timestamp":1751241614000},"source":"Crossref","is-referenced-by-count":0,"title":["The role of the L421P mutation in Penicillin-Binding Protein 1 (PBP1) in the evolution of chromosomally mediated penicillin resistance in\n                  <i>Neisseria gonorrhoeae<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8809-0260","authenticated-orcid":false,"given":"Gabriella","family":"Gentile","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2711-9533","authenticated-orcid":false,"given":"Bryan","family":"Guzman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8209-9520","authenticated-orcid":false,"given":"Adriana","family":"Le Van","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5118-3172","authenticated-orcid":false,"given":"Ann E.","family":"Jerse","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5646-1314","authenticated-orcid":false,"given":"Yonatan H.","family":"Grad","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel","family":"Dominguez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6255-690X","authenticated-orcid":false,"given":"Tatum D.","family":"Mortimer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2507-8974","authenticated-orcid":false,"given":"Robert A.","family":"Nicholas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025070417251964000_2025.06.27.662027v2.1","unstructured":"World Health Organization. 2024. 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