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Mammalian RhoA was the target substrate for its transferase activity suggesting that it may be the biological target of C3larvinA. The kinetic parameters of the NAD+ substrate for the transferase (KM = 75 \u00b1 10 \u00b5M) and glycohydrolase (GH) (KM = 107 \u00b1 20 \u00b5M) reactions were typical for a C3-like bacterial toxin, including the Plx2A virulence factor from Paenibacillus larvae ERIC I. Upon cytoplasmic expression in yeast, C3larvinA caused a growth-defective phenotype indicating that it is an active C3-like toxin and is cytotoxic to eukaryotic cells. The catalytic variant of the Q187-X-E189 motif in C3larvinA showed no cytotoxicity toward yeast confirming that the cytotoxicity of this factor depends on its enzymatic activity. A homology consensus model of C3larvinA with NAD+ substrate was built on the structure of Plx2A, provided additional confirmation that C3larvinA is a member of the C3-like mono-ADP-ribosylating toxin subgroup. A homology model of C3larvinA with NADH and RhoA was built on the structure of the C3cer-NADH-RhoA complex which provided further evidence that C3larvinA is a C3-like toxin that shares an identical catalytic mechanism with C3cer from Bacillus cereus. C3larvinA induced actin cytoskeleton reorganization in murine macrophages, whereas in insect cells, vacuolization and bi-nucleated cells were observed. These cellular effects are consistent with C3larvinA disrupting RhoA function by covalent modification that is shared among C3-like bacterial toxins.<\/jats:p>","DOI":"10.1042\/bsr20193405","type":"journal-article","created":{"date-parts":[[2020,1,10]],"date-time":"2020-01-10T13:09:28Z","timestamp":1578661768000},"update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Characterization of C3larvinA, a novel RhoA-targeting ADP-ribosyltransferase toxin produced by the honey bee pathogen,<i>Paenibacillus larvae<\/i>"],"prefix":"10.1042","volume":"40","author":[{"given":"Madison","family":"Turner","sequence":"first","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Olivier","family":"Tremblay","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kayla\u00a0A.","family":"Heney","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miguel\u00a0R.","family":"Lugo","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julia","family":"Ebeling","sequence":"additional","affiliation":[{"name":"Institute for Bee Research, Department of Molecular Microbiology and Bee Diseases, Hohen Neuendorf 16540, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elke","family":"Genersch","sequence":"additional","affiliation":[{"name":"Institute for Bee Research, Department of Molecular Microbiology and Bee Diseases, Hohen Neuendorf 16540, Germany"},{"name":"Freie Universit\u00e4t Berlin, Fachbereich Veterin\u00e4rmedizin, Institut f\u00fcr Mikrobiologie und Tierseuchen, Berlin 14163, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8559-8384","authenticated-orcid":false,"given":"A.\u00a0Rod","family":"Merrill","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","published-online":{"date-parts":[[2020,1,10]]},"reference":[{"key":"2021111619580056400_B1","doi-asserted-by":"publisher","first-page":"1608","DOI":"10.1126\/science.1230200","article-title":"Wild pollinators enhance fruit set of crops regardless of honey bee abundance","volume":"339","author":"Garibaldi","year":"2013","journal-title":"Science"},{"key":"2021111619580056400_B2","doi-asserted-by":"publisher","first-page":"303","DOI":"10.1098\/rspb.2006.3721","article-title":"Importance of pollinators in changing landscapes for world crops","volume":"274","author":"Klein","year":"2007","journal-title":"Proc. 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