{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:57:25Z","timestamp":1782280645596,"version":"3.54.5"},"posted":{"date-parts":[[2025,7,10]]},"group-title":"Microbiology","reference-count":54,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,10]],"date-time":"2025-07-10T00:00:00Z","timestamp":1752105600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000072","name":"NIH NIDCR","doi-asserted-by":"crossref","award":["R01 DE032555"],"award-info":[{"award-number":["R01 DE032555"]}],"id":[{"id":"10.13039\/100000072","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIH-GMS","award":["R35 GM153272"],"award-info":[{"award-number":["R35 GM153272"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant\n                  <jats:italic>Staphylococcus aureus<\/jats:italic>\n                  by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of\n                  <jats:italic>Streptococcus mutans<\/jats:italic>\n                  , this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited\n                  <jats:italic>S. mutans<\/jats:italic>\n                  growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29 but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including\n                  <jats:italic>Enterococcus faecalis<\/jats:italic>\n                  and\n                  <jats:italic>S. aureus<\/jats:italic>\n                  . Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. At the same time, exposure to HP-29 led to a slight increase in intracellular zinc, mirroring the increase observed in cells exposed to excess zinc. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by \u223c50%. Transcriptome analysis showed that HP-29, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    Widespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen\n                    <jats:italic>Streptococcus mutans<\/jats:italic>\n                    and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29\u2019s efficacy was also observed in other Gram-positive pathogens, including\n                    <jats:italic>Enterococcus faecalis<\/jats:italic>\n                    and\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    . Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when\n                    <jats:italic>S. mutans<\/jats:italic>\n                    is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.07.10.664208","type":"posted-content","created":{"date-parts":[[2025,7,11]],"date-time":"2025-07-11T04:00:28Z","timestamp":1752206428000},"source":"Crossref","is-referenced-by-count":0,"title":["Zinc-Enhanced Activity of an Antimicrobial Halogenated Phenazine Against\n                  <i>Streptococcus mutans<\/i>\n                  and Other Gram-positive Bacteria"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8977-8449","authenticated-orcid":false,"given":"Jessica K.","family":"Kajfasz","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah B.","family":"Hosay","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5043-9669","authenticated-orcid":false,"given":"Qiwen","family":"Gao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3811-2721","authenticated-orcid":false,"suffix":"III","given":"Robert W.","family":"Huigens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9 A.","family":"Lemos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025071208300822000_2025.07.10.664208v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/s0140-6736(21)02724-0"},{"key":"2025071208300822000_2025.07.10.664208v1.2","doi-asserted-by":"publisher","DOI":"10.1007\/s10096-025-05126-4"},{"key":"2025071208300822000_2025.07.10.664208v1.3","doi-asserted-by":"crossref","first-page":"1472","DOI":"10.1039\/D3MD00204G","article-title":"Design, synthesis and evaluation of halogenated phenazine antibacterial prodrugs targeting nitroreductase enzymes for activation","volume":"14","year":"2023","journal-title":"RSC Med Chem"},{"key":"2025071208300822000_2025.07.10.664208v1.4","doi-asserted-by":"publisher","DOI":"10.1099\/00222615-34-4-213"},{"key":"2025071208300822000_2025.07.10.664208v1.5","doi-asserted-by":"crossref","first-page":"14819","DOI":"10.1002\/anie.201508155","article-title":"Halogenated Phenazines that Potently Eradicate Biofilms, MRSA Persister Cells in Non-Biofilm Cultures, and Mycobacterium tuberculosis","volume":"54","year":"2015","journal-title":"Angew Chem Int Ed Engl"},{"key":"2025071208300822000_2025.07.10.664208v1.6","doi-asserted-by":"crossref","first-page":"3962","DOI":"10.1021\/acs.jmedchem.7b01903","article-title":"An Efficient Buchwald-Hartwig\/Reductive Cyclization for the Scaffold Diversification of Halogenated Phenazines: Potent Antibacterial Targeting, Biofilm Eradication, and Prodrug Exploration","volume":"61","year":"2018","journal-title":"J Med Chem"},{"key":"2025071208300822000_2025.07.10.664208v1.7","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.5b02004"},{"key":"2025071208300822000_2025.07.10.664208v1.8","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1021\/acsinfecdis.1c00402","article-title":"Modular Synthetic Routes to Fluorine-Containing Halogenated Phenazine and Acridine Agents That Induce Rapid Iron Starvation in Methicillin-Resistant Staphylococcus aureus Biofilms","volume":"8","year":"2022","journal-title":"ACS Infect Dis"},{"key":"2025071208300822000_2025.07.10.664208v1.9","doi-asserted-by":"crossref","first-page":"2003","DOI":"10.1038\/s41598-017-01045-3","article-title":"A Highly Potent Class of Halogenated Phenazine Antibacterial and Biofilm-Eradicating Agents Accessed Through a Modular Wohl-Aue Synthesis","volume":"7","year":"2017","journal-title":"Sci Rep"},{"key":"2025071208300822000_2025.07.10.664208v1.10","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.1c00168"},{"key":"2025071208300822000_2025.07.10.664208v1.11","doi-asserted-by":"crossref","first-page":"15523","DOI":"10.1002\/anie.201809785","article-title":"Transcript Profiling of MRSA Biofilms Treated with a Halogenated Phenazine Eradicating Agent: A Platform for Defining Cellular Targets and Pathways Critical to Biofilm Survival","volume":"57","year":"2018","journal-title":"Angew Chem Int Ed Engl"},{"key":"2025071208300822000_2025.07.10.664208v1.12","doi-asserted-by":"crossref","unstructured":"Abranches J , Zeng L , Kajfasz JK , Palmer SR , Chakraborty B , Wen ZT , Richards VP , Brady LJ , Lemos JA . 2018. 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