{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T01:50:29Z","timestamp":1768528229082,"version":"3.49.0"},"posted":{"date-parts":[[2025,10,11]]},"group-title":"Microbiology","reference-count":27,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:00:00Z","timestamp":1760140800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Bacillus anthracis<\/jats:italic>\n                  is a gram-positive spore-forming bacterium that causes lethal inhalation anthrax. The use of\n                  <jats:italic>B. anthracis<\/jats:italic>\n                  as a bioweapon is predicated in its ability to form dormant and resistant infective spores that can be used as agents.\n                  <jats:italic>B. anthracis<\/jats:italic>\n                  spores contain large concentrations of dipicolinic acid complexed with cations, especially calcium (Ca-DPA). Following phagocytosis by alveolar macrophages, spores germinate inside the phagolysosome and excrete the Ca-DPA depot into the phagosomal space. In this study, we assessed the effects of DPA on\n                  <jats:italic>B. anthracis<\/jats:italic>\n                  spore biology and cytotoxicity. We generated\n                  <jats:italic>B. anthracis<\/jats:italic>\n                  mutants with defects in DPA synthesis (\n                  <jats:italic>\u0394spoVFA, \u0394spoVFB, \u0394spoVFAB<\/jats:italic>\n                  ) or transport (\n                  <jats:italic>\u0394spoVV<\/jats:italic>\n                  ). To increase the viability of DPA-less spores, we also constructed double mutants (\n                  <jats:italic>\u0394sleB\u0394spoVFA, \u0394sleB\u0394spoVFB, \u0394sleB\u0394spoVFAB<\/jats:italic>\n                  and\n                  <jats:italic>\u0394sleB\u0394spoVV<\/jats:italic>\n                  ) by deleting the cortex lytic\n                  <jats:italic>sleB<\/jats:italic>\n                  gene. We found that single- and double-mutant DPA-less spores were profoundly compromised in dormancy, viability, germination, and heat resistance. Contrary to expectations, each DPA synthesis mutant exhibited distinct viability and resistance phenotypes. Even with compromised stability, DPA-less\n                  <jats:italic>B. anthracis<\/jats:italic>\n                  spores, with the exception of the \u0394\n                  <jats:italic>sleB<\/jats:italic>\n                  \u0394\n                  <jats:italic>spoVV<\/jats:italic>\n                  double mutant, were as cytotoxic as wild-type spores. In summary, DPA is required to sustain normal\n                  <jats:italic>B. anthracis<\/jats:italic>\n                  spore biology but is not required for macrophage-targeted virulence. Furthermore, the SpoVV transporter and SleB lytic protein seem to have redundant roles in anthrax spore cytotoxicity beyond DPA accumulation.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Bacillus anthracis<\/jats:italic>\n                    causes deadly pulmonary anthrax and has been used as a weapon for bioterrorism.\n                    <jats:italic>B. anthracis<\/jats:italic>\n                    form spores that germinate and establish infection. During germination,\n                    <jats:italic>B. anthracis<\/jats:italic>\n                    spores release large amounts of calcium complexed with dipicolinic acid (DPA). In this study, we deleted\n                    <jats:italic>the B. anthracis<\/jats:italic>\n                    genes that are required to synthesize and transport DPA into spores. We found that\n                    <jats:italic>B. anthracis<\/jats:italic>\n                    DPA-less mutant spores exhibited differential biological effects that were DPA independent. Furthermore, we found that DPA was not required for anthrax cytotoxicity. Finally, we found that proteins involved in DPA synthesis, transport, and cortex lysis have biological and virulence functions that extend beyond DPA accumulation.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.10.10.681759","type":"posted-content","created":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T06:25:11Z","timestamp":1760250311000},"source":"Crossref","is-referenced-by-count":0,"title":["Effects of dipicolinic acid on\n                  <i>Bacillus anthracis<\/i>\n                  spore biology and cytotoxicity"],"prefix":"10.64898","author":[{"given":"Chandler","family":"Hassan-Casarez","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tiffany V.","family":"Mata","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2628-9493","authenticated-orcid":false,"given":"Ernesto","family":"Abel-Santos","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025101504000452000_2025.10.10.681759v1.1","doi-asserted-by":"crossref","unstructured":"Ehling-Schulz M , Lereclus D , Koehler TM . 2019. The Bacillus cereus group: Bacillus species with pathogenic potential. Microbiol Spectr 7.","DOI":"10.1128\/microbiolspec.GPP3-0032-2018"},{"key":"2025101504000452000_2025.10.10.681759v1.2","doi-asserted-by":"crossref","first-page":"20","DOI":"10.12998\/wjcc.v3.i1.20","article-title":"Anthrax: A disease of biowarfare and public health importance","volume":"3","year":"2015","journal-title":"World J Clin Cases"},{"key":"2025101504000452000_2025.10.10.681759v1.3","unstructured":"Simonsen KA , Chatterjee K . 2022. Anthrax. 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Improvements to a markerless allelic exchange system for Bacillus anthracis. PLoS One 10.","DOI":"10.1371\/journal.pone.0142758"},{"key":"2025101504000452000_2025.10.10.681759v1.22","doi-asserted-by":"publisher","DOI":"10.1111\/jam.14067"},{"key":"2025101504000452000_2025.10.10.681759v1.23","unstructured":"R Core Team . 2025. R: A Language and Environment for Statistical Computing. v4.4.3. R Foundation for Statistical Computing, Vienna."},{"key":"2025101504000452000_2025.10.10.681759v1.24","doi-asserted-by":"publisher","DOI":"10.1128\/JB.05126-11"},{"key":"2025101504000452000_2025.10.10.681759v1.25","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M611432200"},{"key":"2025101504000452000_2025.10.10.681759v1.26","doi-asserted-by":"crossref","unstructured":"Setlow P . 2014. Spore resistance properties. 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