Abstract
Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.
| Original language | English |
|---|---|
| Pages (from-to) | 823-828 |
| Number of pages | 6 |
| Journal | Journal of Veterinary Medical Science |
| Volume | 77 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - Aug 1 2015 |
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Keywords
- Foodborne disease
- Signature-tagged mutagenesis
- Vaccine
- Vibrio vulnificus
- Virulence
ASJC Scopus subject areas
- veterinary(all)
Cite this
Signature-tagged mutagenesis of vibrio vulnificus. / Yamamoto, Mai; Kashimoto, Takashige; Tong, Ping; Xiao, Jianbo; Sugiyama, Michiko; Inoue, Miyuki; Matsunaga, Rie; Hosohara, Kohei; Nakata, Kazue; Yokota, Kenji; Oguma, Keiji; Yamamoto, Koichiro.
In: Journal of Veterinary Medical Science, Vol. 77, No. 7, 01.08.2015, p. 823-828.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Signature-tagged mutagenesis of vibrio vulnificus
AU - Yamamoto, Mai
AU - Kashimoto, Takashige
AU - Tong, Ping
AU - Xiao, Jianbo
AU - Sugiyama, Michiko
AU - Inoue, Miyuki
AU - Matsunaga, Rie
AU - Hosohara, Kohei
AU - Nakata, Kazue
AU - Yokota, Kenji
AU - Oguma, Keiji
AU - Yamamoto, Koichiro
PY - 2015/8/1
Y1 - 2015/8/1
N2 - Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.
AB - Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.
KW - Foodborne disease
KW - Signature-tagged mutagenesis
KW - Vaccine
KW - Vibrio vulnificus
KW - Virulence
UR - http://www.scopus.com/inward/record.url?scp=84938540169&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84938540169&partnerID=8YFLogxK
U2 - 10.1292/jvms.14-0655
DO - 10.1292/jvms.14-0655
M3 - Article
VL - 77
SP - 823
EP - 828
JO - Journal of Veterinary Medical Science
JF - Journal of Veterinary Medical Science
SN - 0916-7250
IS - 7
ER -