| Kazuhiro Iiyama | Last modified date：2013.4.17 |
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092-642-3033
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092-642-4421
Academic Degree
PhD
Field of Specialization
Biological control, Insect pathology, Bacteriology
Outline Activities
Pseudomonas aeruginosa is an opportunistic pathogen of insects and possesses a manganese- and an iron- superoxide dismutases (SODs). To ascertain the role of these SODs in virulence to the silkworm, Bombyx mori, we have generated three mutants carrying an interrupted sodM (encoding manganese- SOD, Mn-SOD) or sodB (encoding iron- SOD, Fe-SOD) and a sodM sodB double mutation. Total SOD activities of sodM, sodB and sodM sodB mutants in Luria broth and tryptic soy broth were ca. 100%, 0% and 0% of the activity of a wild-type strain, respectively. Whereas the relative activities in low phosphate-succinate broth were 0%, 96% and 0%, respectively. This result indicates that the two SODs of P. aeruginosa are highly regulated by the environmental conditions. To investigate their virulence to the silkworm, the fourth instar larvae were inoculated with various doses of the wild-type and three mutants. The virulence remarkably differed among these strains. The sodM mutant was indistinguishable from the wild-type in aspect of virulence at all inoculation levels (103-106 cells / larva). Whereas the virulence of sodB mutant reduced, when relatively low cells number (103-104) was inoculated. Moreover, the double mutant could not cause the any fatal damage at 105 cells or lower. These results suggest that the SODs are required for full virulence of P. aeruginosa to the silkworm and Fe-SOD is more important than Mn-SOD in the infection process.
Ribosomal DNA (rDNA) containing small subunit (SSU) rDNA and both flanking regions in the entomopathogenic microsporidian Nosema bombycis NIS 001 was amplified from genomic DNA with a primer set based on the sequence of an inverse polymerase chain reaction (PCR) -derived fragment. In this fragment, SSU rDNA was divided by a 618-bp insert at nt 599, and 5S rDNA was located downstream of the SSU rDNA, fragmented by 284-bp intergenic spacer. In addition, the 48-bp 3' end of large subunit (LSU) rDNA was located 118 bp upstream of the fragmented SSU rDNA. In the amplicon, the region upstream of the LSU rDNA was a homologue of the C-terminal CHARLIE8 transposon-like element of human GTF2IRD2. In this organism, another fragmented SSU rDNA, which was divided by a 231-bp insert at nt 50, was also detected. Both the intact (insertless) and fragmented SSU rDNAs clustered with LSU rDNA and 5S rDNA and the intergenic sequences between SSU rDNA and 5S rDNA were divergent in an organism. Reverse transcription (RT) - PCR assay indicated that not only the intact SSU rDNA but also the fragmened SSU rDNA were transcribed in N. bombycis.
Ribosomal DNA (rDNA) containing small subunit (SSU) rDNA and both flanking regions in the entomopathogenic microsporidian Nosema bombycis NIS 001 was amplified from genomic DNA with a primer set based on the sequence of an inverse polymerase chain reaction (PCR) -derived fragment. In this fragment, SSU rDNA was divided by a 618-bp insert at nt 599, and 5S rDNA was located downstream of the SSU rDNA, fragmented by 284-bp intergenic spacer. In addition, the 48-bp 3' end of large subunit (LSU) rDNA was located 118 bp upstream of the fragmented SSU rDNA. In the amplicon, the region upstream of the LSU rDNA was a homologue of the C-terminal CHARLIE8 transposon-like element of human GTF2IRD2. In this organism, another fragmented SSU rDNA, which was divided by a 231-bp insert at nt 50, was also detected. Both the intact (insertless) and fragmented SSU rDNAs clustered with LSU rDNA and 5S rDNA and the intergenic sequences between SSU rDNA and 5S rDNA were divergent in an organism. Reverse transcription (RT) - PCR assay indicated that not only the intact SSU rDNA but also the fragmened SSU rDNA were transcribed in N. bombycis.
Research
Research Interests
- Study on phospholipases produced by Pseudomonas aeruginosa
keyword : Pseudomonas aeruginosa, Bombyx mori, phospholipase C, plcB, plcH, plcN
2006.04. - Study on superoxide dismutases produced by Pseudomonas aeruginosa
keyword : Pseudomonas aeruginosa, Bombyx mori, virulence，superoxide dismutase，sodM, sodB
2004.04. - Study on the toxic protein produced by the bacteria isolated from insect
keyword : entomopathogenic bacteria, bacterial isolates from ant lion, virulence, virulence factor
2003.04. - Study on a two component, GacS-GacA in an oppotunistic bacteria Pseudomonas aeruginosa
keyword : entomopathogenic bacteria, two component system
2001.04. - Ribosomal DNA (rDNA) containing small subunit (SSU) rDNA and both flanking regions in the entomopathogenic microsporidian Nosema bombycis NIS 001 was amplified from genomic DNA with a primer set based on the sequence of an inverse polymerase chain reaction (PCR) -derived fragment. In this fragment, SSU rDNA was divided by a 618-bp insert at nt 599, and 5S rDNA was located downstream of the SSU rDNA, fragmented by 284-bp intergenic spacer. In addition, the 48-bp 3' end of large subunit (LSU) rDNA was located 118 bp upstream of the fragmented SSU rDNA. In the amplicon, the region upstream of the LSU rDNA was a homologue of the C-terminal CHARLIE8 transposon-like element of human GTF2IRD2. In this organism, another fragmented SSU rDNA, which was divided by a 231-bp insert at nt 50, was also detected. Both the intact (insertless) and fragmented SSU rDNAs clustered with LSU rDNA and 5S rDNA and the intergenic sequences between SSU rDNA and 5S rDNA were divergent in an organism. Reverse transcription (RT) - PCR assay indicated that not only the intact SSU rDNA but also the fragmened SSU rDNA were transcribed in N. bombycis.
Papers
| 1. | Arai Hiroyuki, Iiyama Kazuhiro,Role of nitric oxide-detoxifying enzymes in virulence of Pseudomonas aeruginosaagainst the silkworm, Bombyx mori,Biosci. Biotechnol. Biochem,Vol.77,No.1,2013.01. |
| 2. | Kaibara Fusako, Iiyama Kazuhiro, Chieda Yuuka, Lee Jae Man, kusakabe takahiro, Yasunaga-Aoki Chisa, Shimizu Susumu,Construction of serralysin-like metalloprotease-deficient mutants of Serratia liquefaciens and their virulence in the silkworm, Bombyx mori,J. Insect Biotechnol. Sericol. ,Vol.81,2012.12. |
| 3. | Chieda, Y., Iiyama, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S.,Virulence of an exotoxin A-deficient strain of Pseudomonas aeruginosa toward the silkworm, Bombyx mori.,Microbial Pathogenesis,Vol.51,No.6,407-414,2011.12. |
| 4. | Iiyama, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S. ,Improvement in pHERD vectors to express recombinant proteins tagged with hexahistidine at either the NH2- or COOH- terminal in Pseudomonas aeruginosa,J. Insect Biotechnol. Sericol.,Vol.80,57-61,2011.06. |
| 5. | Egami, I., Iiyama, K., Zhang, P., Chieda, Y., Ino, N., Hasegawa, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S.,Insecticidal bacterium isolated from an ant lion larva from Munakata, Japan.,J. Appl. Entomol.,vol. 133 No. 2 117-124,2009.03. |
| 6. | Iiyama, K., Chieda, Y., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S.,Virulence of Phospholipase C Mutants of Pseudomonas aeruginosa PAO1 against the Silkworm, Bombyx mori.,J. Insect Biotechnol. Sericol.,vol. 77, No. 2, 115-120,2008.06. |
| 7. | Chieda, Y., Iiyama, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S.,Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori,FEMS Microbiology Letters,278 101-107,2008.01. |
| 8. | Chieda, Y., Iiyama, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S.,The gacA gene of Pseudomonas aeruginosa PAO1 is not required for full virulence in Bombyx mori.,J. Insect Biotechnol. Sericol. ,Vol. 76, 89-95,2007.06. |
| 9. | Iiyama K, Chieda Y, Lee JM, Kusakabe T, Yasunaga-Aoki C, Shimizu S.,Effect of superoxide dismutase gene inactivation on virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori,Appl Environ Microbiol.,73(5):1569-75,2007.03. |
| 10. | Chieda, Y., Iiyama, K., Yasunaga-Aoki, C., Lee, J.M., Kusakabe, T. and Shimizu, S.,Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori,FEMS Microbiol. Lett.,Vol.244,No.1,244巻 181-186,2005.03. |
| 11. | Iiyama, K., Chieda, Y., Yasunaga-Aoki, C., Hayasaka, S. and Shimizu, S.,Analyses of the ribosomal DNA region in Nosema bombycis NIS 001.,J. Eukaryot. Microbiol,Vol.51,No.6,51巻 598-604.,2004.01. |
Presentations
| 1. | Pseudomonas aeruginosa is a Gram-negative, rod shaped bacterium and human opportunistic pathogen. Since several strains of the P. aeruginosa infect plants, nematodes and insects, some organisms have been used as model hosts of P. aeruginosa infection. To use silkworm, Bombyx mori, as a model host, it is important to compare the bacterial virulence strategies between the different hosts, mammal and B. mori. We created the mutants of GacS-GacA two component system (gacA), pyocyanin production (phzM, phzS), superoxide dismutase (SOD) production (sodM, sodB, sodM sodB) and nucleotide excision repair (NER) mechanism (uvrC) originated from P. aeruginosa PAO1. Among these genes, gacA, sodB phzM and phzS have been reported to be important in the virulence to mammalian hosts. Gene disruption in each mutant was confirmed by Southern hybridization and change of phenotypic characteristics (pyocyanin production, SOD activity and UV sensitivity). Inoculation test demonstrated that gacA, phzM, phzS mutants had the same level of virulence in comparison with the parent strain, PAO1. Whereas, sodB, sodM sodB and uvrC mutants showed reduced virulence. In addition, the virulence of the mutants was restored by the complementation of the corresponding genes in trans. These results suggested that the importance of these virulence factors depended on the host species, but that of the survival factors were common in P. aeruginosa.. |
| 2. | It is known that the GacS/GacA two-component system is important for the virulence of Pseudomonas aeruginosa in invertebrate and vertebrate hosts and in plants. In this study, the gacA nonpolar mutant C2 and polar mutant C3 were generated and inoculated into Bombyx mori. The polar mutants showed reduced virulence, whereas no significant difference was observed between the gacA nonpolar mutant and wild type strain with respect to the ability to kill B. mori larvae. This suggests that the reduced virulence was due to a polar effect of the insertions, rather than to the lack of the gacA gene product. In the genome, the gacA gene is directly followed by a uvrC gene. To clarify the importance of uvrC to virulence, the uvrC mutant strain UC was constructed. UC strains were assessed for the production of virulence factors (pyocyanin, pyoverdine, elastase and total protease) in comparison to UC strains. No difference was observed between PAO1 (parent strain) and UC strains, suggesting that the mutation of uvrC had no effect on the production of these virulence factors. UvrC is an endonuclease involved in NER, which is one of the excision repair systems. We tested the survival of UC strains exposed to cisplatin or UV. The survival rate of UC (pBBR1MCS2) after cisplatin or UV treatment was clearly lower than that of PAO1 (pBBR1MCS2). To investigate the influence of uvrC on virulence of hemocoel infection, UC strains were injected into B. mori. UC was shown to be less virulent than PAO1. This result suggests that not gacA rather than uvrC is necessary for the full virulence of P. aeruginosa PAO1 to in B. mori.. |
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Study assistance about the characterization, infection / multiplication style, a host and a parasite interaction, insect pathology, and insect microbiology.
The instruction assistance of an experiment and advice which are performed with the latest technology.
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