{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T01:32:56Z","timestamp":1787016776176,"version":"build-2736575974"},"reference-count":38,"publisher":"American Society for Microbiology","issue":"20","license":[{"start":{"date-parts":[[2016,9,30]],"date-time":"2016-09-30T00:00:00Z","timestamp":1475193600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2016,10,15]],"date-time":"2016-10-15T00:00:00Z","timestamp":1476489600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"HHS | National Institutes of Health","doi-asserted-by":"publisher","award":["R01 AI098833-01"],"award-info":[{"award-number":["R01 AI098833-01"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009591","name":"North Carolina Agricultural Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100009591","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2016,10,15]]},"abstract":"ABSTRACT<\/jats:title>\n \n Clostridium botulinum<\/jats:named-content>\n and\n Bacillus anthracis<\/jats:named-content>\n produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of\n Lactobacillus acidophilus<\/jats:named-content>\n NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of\n Clostridium botulinum<\/jats:named-content>\n serotype A neurotoxin (BoNT\/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT\/A-Hc and the PA. The BoNT\/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from\n L. acidophilus<\/jats:named-content>\n and a dendritic-cell-targeting peptide. A chromosomal region downstream of\n lba0889<\/jats:italic>\n carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids.\n <\/jats:p>\n \n IMPORTANCE<\/jats:bold>\n Clostridium botulinum<\/jats:named-content>\n and\n Bacillus anthracis<\/jats:named-content>\n produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid-based expression systems since expressing antigens from a chromosomal location confers an advantage to the vaccine strains by eliminating the antibiotic maintenance required for plasmids and negates issues with plasmid instability that would result in loss of the antigen. Lactic acid bacteria, including\n Lactobacillus acidophilus<\/jats:named-content>\n , have shown potential for mucosal vaccine delivery, as\n L. acidophilus<\/jats:named-content>\n is bile and acid tolerant, allowing transit through the gastrointestinal tract where cells interact with host epithelial and immune cells, including dendritic cells. In this study, we successfully expressed\n C. botulinum<\/jats:named-content>\n and\n B. anthracis<\/jats:named-content>\n antigens in the probiotic\n L. acidophilus<\/jats:named-content>\n strain NCFM. Both antigens were highly expressed individually or in tandem from the chromosome of\n L. acidophilus<\/jats:named-content>\n .\n <\/jats:p>","DOI":"10.1128\/aem.01533-16","type":"journal-article","created":{"date-parts":[[2016,8,5]],"date-time":"2016-08-05T22:48:40Z","timestamp":1470437320000},"page":"6091-6101","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":31,"title":["Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus"],"prefix":"10.1128","volume":"82","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8629-0675","authenticated-orcid":false,"given":"Sarah","family":"O'Flaherty","sequence":"first","affiliation":[{"name":"Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Todd R.","family":"Klaenhammer","sequence":"additional","affiliation":[{"name":"Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro1840"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.3168\/jds.S0022-0302(01)74481-5"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0810305105"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0900029106"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.172.12.7425"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.05153-11"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/CVI.00049-12"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-197-0_22"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.06028-11"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1128\/mr.46.1.86-94.1982"},{"key":"e_1_3_3_12_2","first-page":"1454","article-title":"Notice of CDC's discontinuation of investigational pentavalent (ABCDE) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins","volume":"60","author":"Centers for Disease Control and Prevention","year":"2011","unstructured":"Centers for Disease Control and Prevention. 2011. 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