{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T10:38:47Z","timestamp":1790419127450,"version":"4.1.0"},"reference-count":27,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2017,6,15]],"date-time":"2017-06-15T00:00:00Z","timestamp":1497484800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs), toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC), alternating in size from 300 kDa to 500 kDa. These progenitor complexes can be classified into hemagglutinin positive or hemagglutinin negative, depending on the ability of some of the neurotoxin-associated proteins (NAPs) to cause hemagglutination. The hemagglutinin positive progenitor toxin complex consists of BoNT, nontoxic non-hemagglutinin (NTNH), and three hemagglutinin proteins; HA-70, HA-33, and HA-17. Hemagglutinin negative progenitor toxin complexes contain BoNT and NTNH as the minimally functional PTC (M-PTC), but not the three hemagglutinin proteins. Interestingly, the genome of hemagglutinin negative progenitor toxin complexes comprises open reading frames (orfs) which encode for three proteins, but the existence of these proteins has not yet been extensively demonstrated. In this work, we demonstrate that these three proteins exist and form part of the PTC for hemagglutinin negative complexes. Several hemagglutinin negative strains producing BoNT\/A, \/E, and \/F were found to contain the three open reading frame proteins. Additionally, several BoNT\/A-containing bivalent strains were examined, and NAPs from both genes, including the open reading frame proteins, were associated with BoNT\/A. The open reading frame encoded proteins are more easily removed from the botulinum complex than the hemagglutinin proteins, but are present in several BoNT\/A and \/F toxin preparations. These are not easily removed from the BoNT\/E complex, however, and are present even in commercially-available purified BoNT\/E complex.<\/jats:p>","DOI":"10.3390\/toxins9060193","type":"journal-article","created":{"date-parts":[[2017,6,15]],"date-time":"2017-06-15T10:07:33Z","timestamp":1497521253000},"page":"193","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Characterization of Hemagglutinin Negative Botulinum Progenitor Toxins"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8067-136X","authenticated-orcid":false,"given":"Suzanne","family":"Kalb","sequence":"first","affiliation":[{"name":"Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jakub","family":"Baudys","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Theresa","family":"Smith","sequence":"additional","affiliation":[{"name":"Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Ft. Detrick, MD 21702, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonard","family":"Smith","sequence":"additional","affiliation":[{"name":"Office of the Chief Scientist, Medical Research and Materiel Command (MRMC), Fort Detrick, MD 21702, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Barr","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2017,6,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"910","DOI":"10.1128\/iai.16.3.910-914.1977","article-title":"Correlation between oral toxicity and in vitro stability of Clostridium botulinum type A and B toxins of different molecular sizes","volume":"16","author":"Sugii","year":"1977","journal-title":"Infect. Immun."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"6255","DOI":"10.1038\/ncomms7255","article-title":"Botulinum toxin a complex exploits intestinal m cells to enter the host and exert neurotoxicity","volume":"6","author":"Matsumura","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1083\/jcb.200910119","article-title":"Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin","volume":"189","author":"Sugawara","year":"2010","journal-title":"J. Cell Biol"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"977","DOI":"10.1126\/science.1214270","article-title":"Botulinum neurotoxin is shielded by NTNHA in an interlocked complex","volume":"335","author":"Gu","year":"2012","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/S0014-5793(00)01147-9","article-title":"Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes","volume":"467","author":"Fujinaga","year":"2000","journal-title":"FEBS Lett."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Lee, K., Gu, S., Jin, L., Le, T.T., Cheng, L.W., Strotmeier, J., Kruel, A.M., Yao, G., Perry, K., and Rummel, A. (2013). Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003690"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1126\/science.1253823","article-title":"Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin a complex","volume":"344","author":"Lee","year":"2014","journal-title":"Science"},{"key":"ref_8","first-page":"238","article-title":"Comparative antigenicity of three preparations of botulium neurotoxin type A in the rabbit","volume":"9","author":"Frevert","year":"2006","journal-title":"Neurotox. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1016\/j.toxicon.2009.01.017","article-title":"Immunological characterization of the subunits of type A botulinum neurotoxin and different components of its associated proteins","volume":"53","author":"Kukreja","year":"2009","journal-title":"Toxicon"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3739","DOI":"10.1099\/mic.0.28421-0","article-title":"Production of anti-neurotoxin antibody is enhanced by two subcomponents, HA1 and HA3b, of Clostridium botulinum type B 16s toxin-haemagglutinin","volume":"151","author":"Lee","year":"2005","journal-title":"Microbiology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.tox.2008.04.018","article-title":"Effects of purification on the bioavailability of botulinum neurotoxin type A","volume":"249","author":"Cheng","year":"2008","journal-title":"Toxicology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1128\/iai.16.1.107-109.1977","article-title":"Oral toxicities of Clostridium botulinum toxins in response to molecular size","volume":"16","author":"Ohishi","year":"1977","journal-title":"Infect. Immun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1046\/j.1365-2958.1998.00985.x","article-title":"BotR\/A is a positive regulator of botulinum neurotoxin and associated non-toxin protein genes in Clostridium botulinum A","volume":"29","author":"Marvaud","year":"1998","journal-title":"Mol. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4478","DOI":"10.1128\/AEM.71.8.4478-4486.2005","article-title":"Characterization of botulinum progenitor toxins by mass spectrometry","volume":"71","author":"Hines","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1128\/AEM.01882-09","article-title":"Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex","volume":"76","author":"Lin","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4091","DOI":"10.1128\/JCM.02392-12","article-title":"Toxin detection in patients\u2019 sera by mass spectrometry during two outbreaks of type A botulism in france","volume":"50","author":"Mazuet","year":"2012","journal-title":"J. Clin. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"14316","DOI":"10.1021\/bi701564f","article-title":"Comparative role of neurotoxin-associated proteins in the structural stability and endopeptidase activity of botulinum neurotoxin complex types A and E","volume":"46","author":"Kukreja","year":"2007","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6140","DOI":"10.1021\/ac0511748","article-title":"Detection of botulinum neurotoxin a in a spiked milk sample with subtype identification through toxin proteomics","volume":"77","author":"Kalb","year":"2005","journal-title":"Anal. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1007\/s00216-012-5767-3","article-title":"De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics","volume":"403","author":"Kalb","year":"2012","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3254","DOI":"10.1021\/ac5001509","article-title":"Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: Bont\/A2, \/F4, and \/F5","volume":"86","author":"Kalb","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1046\/j.1365-2672.1997.00313.x","article-title":"Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins","volume":"84","author":"Collins","year":"1998","journal-title":"J. Appl Microbiol"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.ab.2011.01.025","article-title":"Improved detection of botulinum neurotoxin type A in stool by mass spectrometry","volume":"412","author":"Wang","year":"2011","journal-title":"Anal. Biochem."},{"key":"ref_23","first-page":"27","article-title":"Purification of Clostridium botulinum type A neurotoxin","volume":"Volume 145","author":"Holst","year":"2000","journal-title":"Methods in Molecular Biology"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Hill, K.K., Xie, G., Foley, B.T., Smith, T.J., Munk, A.C., Bruce, D., Smith, L.A., Brettin, T.S., and Detter, J.C. (2009). Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and clostridium butyricum type E strains. BMC Biol., 7.","DOI":"10.1186\/1741-7007-7-66"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Kalb, S.R., Santana, W.I., Geren, I.N., Garcia-Rodriguez, C., Lou, J., Smith, T.J., Marks, J.D., Smith, L.A., Pirkle, J.L., and Barr, J.R. (2011). Extraction and inhibition of enzymatic activity of botulinum neurotoxins \/B1, \/B2, \/B3, \/B4, and \/B5 by a panel of monoclonal anti-BoNT\/B antibodies. BMC Biochem., 12.","DOI":"10.1186\/1471-2091-12-58"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Kalb, S.R., Lou, J., Garcia-Rodriguez, C., Geren, I.N., Smith, T.J., Moura, H., Marks, J.D., Smith, L.A., Pirkle, J.L., and Barr, J.R. (2009). Extraction and inhibition of enzymatic activity of botulinum neurotoxins\/A1, \/A2, and \/A3 by a panel of monoclonal anti-BoNT\/A antibodies. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0005355"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Kalb, S.R., Garcia-Rodriguez, C., Lou, J., Baudys, J., Smith, T.J., Marks, J.D., Smith, L.A., Pirkle, J.L., and Barr, J.R. (2010). Extraction of bont\/A, \/B, \/E, and \/F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by endopep-MS. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0012237"}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/9\/6\/193\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:39:07Z","timestamp":1760207947000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/9\/6\/193"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,6,15]]},"references-count":27,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2017,6]]}},"alternative-id":["toxins9060193"],"URL":"https:\/\/doi.org\/10.3390\/toxins9060193","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,6,15]]}}}