National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki, 305-0856, Japan; Research Institute for Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki, 305-0856, Japan.
Vet Microbiol. 2019 Dec;239:108488. doi: 10.1016/j.vetmic.2019.108488. Epub 2019 Nov 1.
Acriflavine, an acridine dye that causes frameshift mutations, has been used to attenuate various veterinary pathogens for the development of live vaccines. Erysipelothrix rhusiopathiae Koganei 65-0.15 strain (Koganei) (serovar 1a) is the acriflavine-resistant live vaccine currently used in Japan for the control of swine erysipelas. To investigate the attenuation mechanisms of the Koganei strain, we analyzed the draft genome sequence of the Koganei strain against the reference genome sequence of the E. rhusiopathiae Fujisawa strain (serovar 1a). The sequence analysis revealed a high degree of sequence similarity between the two strains and identified a total of 98 sequence differences within 80 protein-coding sequences. Among them, insertions/deletions (indels) were identified in 9 genes, of which 7 resulted in frameshift and premature termination. To investigate whether these mutations resulted in the attenuation of the Koganei strain, we focused on the indel mutation identified in ERH_0661, an XRE family transcriptional regulator. We introduced the mutation into ERH_0661 of the Fujisawa strain and restored the mutation of the Koganei strain. Animal experiments using the recombinant strains showed that mice survived inoculation with 10 colony forming units (CFUs) (equivalent to approximately 100 50% lethal doses [LD50] of the wild-type Fujisawa) of the recombinant Fujisawa strain, and the mice became ill after inoculation with 10 CFUs of the recombinant Koganei strain. These results suggest that the transcriptional regulator ERH_0661 is involved in the virulence of E. rhusiopathiae and that the ERH_0661 mutation is partially responsible for the attenuation of the Koganei strain.
吖啶黄是一种吖啶染料，可引起移码突变，已被用于减毒各种兽医病原体用于开发活疫苗。猪丹毒丝菌科氏 65-0.15 株（科氏）（血清型 1a）是日本目前用于控制猪丹毒的吖啶黄抗性活疫苗。为了研究科氏株的减毒机制，我们针对猪丹毒丝菌藤泽株（血清型 1a）的参考基因组序列对科氏株的草图基因组序列进行了分析。序列分析表明，两株之间具有高度的序列相似性，在 80 个蛋白编码序列中总共鉴定出 98 个序列差异。其中，在 9 个基因中发现了插入/缺失（indels），其中 7 个导致移码和过早终止。为了研究这些突变是否导致科氏株的衰减，我们专注于 ERH_0661 中鉴定出的 indel 突变，ERH_0661 是一个 XRE 家族转录调节因子。我们将突变引入藤泽株的 ERH_0661 中，并恢复了科氏株的突变。使用重组菌株进行的动物实验表明，用 10 个菌落形成单位（CFU）（相当于野生型藤泽株的大约 100 50%致死剂量 [LD50]）的重组藤泽株接种的小鼠存活下来，而用 10 CFU 的重组科氏株接种的小鼠发病。这些结果表明，转录调节因子 ERH_0661 参与了猪丹毒丝菌的毒力，并且 ERH_0661 突变部分导致了科氏株的衰减。
