{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T07:49:30Z","timestamp":1775980170307,"version":"3.50.1"},"posted":{"date-parts":[[2025,3,21]]},"group-title":"Microbiology","reference-count":71,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,21]],"date-time":"2025-03-21T00:00:00Z","timestamp":1742515200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,22]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Bacterial cells are surrounded by a dynamic cell wall which in part is made up of a mesh-like peptidoglycan (PG) layer that provides the cell with structural integrity and resilience. In Gram-positive bacteria, this layer is thick and robust, whereas in Gram-negative bacteria, it is thinner and flexible as the cell is supported by an additional outer membrane. PG undergoes continuous turnover, with degradation products being recycled to maintain cell wall homeostasis. Some Gram-negative species can bypass\n                  <jats:italic>de novo<\/jats:italic>\n                  PG biosynthesis, relying instead on PG recycling to sustain growth and division.\n                  <jats:italic>Legionella pneumophila<\/jats:italic>\n                  (hereafter\n                  <jats:italic>Legionella<\/jats:italic>\n                  ), the causative agent of Legionnaires\u2019 disease, encodes such recycling machinery within its genome. This study investigates the biochemical, genetic, and pathogenic roles of PG recycling in\n                  <jats:italic>Legionella<\/jats:italic>\n                  . Previously, we have shown that PG can be visualized in both model and native systems using a combination of\n                  <jats:italic>N<\/jats:italic>\n                  -acetylmuramic acid (NAM) probes and PG recycling programs. Here, two PG recycling gene homologs in the\n                  <jats:italic>Legionella<\/jats:italic>\n                  genome\n                  <jats:italic>lpg0296<\/jats:italic>\n                  (\n                  <jats:italic>amgK<\/jats:italic>\n                  ) and\n                  <jats:italic>lpg0295<\/jats:italic>\n                  (\n                  <jats:italic>murU)<\/jats:italic>\n                  were identified and characterized; chemical biology strategies were used to rigorously track the incorporation of \u201cclick\u201d-PG-probes. Deletion of\n                  <jats:italic>amgK<\/jats:italic>\n                  abolished PG labeling, while genetic complementation restored labeling. Additionally, copper-free click chemistry with ultra-fast tetrazine-NAM probes enabled live-cell PG labeling. The data suggest that\n                  <jats:italic>amgK<\/jats:italic>\n                  contributes to the pathogenicity of the organism, as\n                  <jats:italic>amgK<\/jats:italic>\n                  deletion increased\n                  <jats:italic>Legionella<\/jats:italic>\n                  \u2019s susceptibility to antibiotics and significantly reduced\n                  <jats:italic>Legionella\u2019<\/jats:italic>\n                  s ability to replicate in host alveolar macrophages. An intracellular replication assay demonstrated that while PG recycling is not essential for internalization, successful replication of\n                  <jats:italic>Legionella<\/jats:italic>\n                  within MH-S murine alveolar macrophages requires functional\n                  <jats:italic>amgK<\/jats:italic>\n                  . These findings underscore the essential role of AmgK in\n                  <jats:italic>Legionella<\/jats:italic>\n                  \u2019s intracellular survival, emphasizing the importance of PG recycling in pathogenicity, and establish a foundation for developing novel\n                  <jats:italic>Legionella<\/jats:italic>\n                  -specific antibiotic strategies.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author summary<\/jats:title>\n                  <jats:p>\n                    In this work, we evaluated the functionality of the peptidoglycan kinase, AmgK, from\n                    <jats:italic>Legionella<\/jats:italic>\n                    . AmgK homologs from other organisms have been shown to play a role in peptidoglycan (PG) recycling. Here we performed a series of chemical biology, genetic and biochemical experiments to show that\n                    <jats:italic>Legionella<\/jats:italic>\n                    carries a functional AmgK. The data show that without AmgK\n                    <jats:italic>Legionella<\/jats:italic>\n                    becomes hypersensitive to Fosfomycin, an antibiotic that targets bacterial cell wall biosynthesis. Moreover, PG recycling is required for\n                    <jats:italic>Legionella<\/jats:italic>\n                    to successfully replicate within a host macrophage. Collectively, the data point to a role of PG recycling in pathogenicity and suggest that inhibiting AmgK could be a powerful strategy in treating Legionnaires\u2019 disease.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.21.644609","type":"posted-content","created":{"date-parts":[[2025,3,22]],"date-time":"2025-03-22T00:03:22Z","timestamp":1742601802000},"source":"Crossref","is-referenced-by-count":1,"title":["The\n                  <i>Legionella pneumophila<\/i>\n                  peptidoglycan recycling kinase, AmgK, is essential for survival and replication inside host alveolar macrophages"],"prefix":"10.64898","author":[{"given":"Sushanta","family":"Ratna","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lina","family":"Pradhan","sequence":"additional","affiliation":[{"name":"Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marina P.","family":"Vasconcelos","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aastha","family":"Acharya","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bella","family":"Carnahan","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alex","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6582-8146","authenticated-orcid":false,"given":"Arit","family":"Ghosh","sequence":"additional","affiliation":[{"name":"Delaware Biotechnology Institute, UD Flow Cytometry & Single Cell Core, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Abigail","family":"Bolt","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jacob","family":"Ellis","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephen N.","family":"Hyland","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ashlyn S.","family":"Hillman","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joseph M.","family":"Fox","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"April","family":"Kloxin","sequence":"additional","affiliation":[{"name":"Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States"},{"name":"Department of Materials Science & Engineering, University of Delaware, Newark, Delaware 19716, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M. 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