| Budget Amount *help |
¥3,730,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥330,000)
Fiscal Year 2007: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2006: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
In recent years, many new staphylococcal enterotoxins (SEs) have been reported. It has been known that certain SE genes are associated with mobile genetic elements such as pathogenicity islands, prophages, and plasmids. These facts imply that superantigenic toxin genes are transferred horizontally between staphylococcal strains. There is a possibility that these mobile genetic elements have played an important role in the evolution of S. aureus as a pathogen. To investigate pathobiology and epidemiology of these newly identified Ses, we had undertaken studies shown in below.
1. To detect and characterize unknown SE gene encoding pathogenicity islands, we have developed a new method, named pathogenicity islands scanning. Based on full genome sequences of several S. aureus, we designed PCR primers to amplify entire region of pathogenicity islands. Amplicons were subjected to restriction fragments length polymorphism (RFLP) analysis. Using pathogenicity islands scanning/RFLP, We have found
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that SEB-related pathogenicity islands are classified into at least 5 groups. Also, we have determined complete nucleotide sequence of selj/selr encoding plasmid pF5 using shotgun sequencing strategy. This plasmid is encoding two novel enterotoxin gene, ses and set, in addition to selj and selr. In addition, we developed several diagnostic methods including immuno-PCR sensitive detection of SEs, cultivation/PCR of sensitive detection of S. aureus, and multiplex PCR for comprehensive detection of SE/SE1 genes.
2. We characterized two novel staphylococcal entertoxins SES and SET. Recombinant (r)SES specifically stimulated human T cells in T cell receptor (TCR) Vβ9-specific manner in the presence of MHC class II^+ antigen presenting cells (APCs). rSET also stimulated T cells in the presence of MHC class II^+ APC, although its Vβ skewing was not found in reactive T cells. Subsequently, we examined the emetic activity of SES and SET. We also studied SEIR to determine emetic activity in primates. This toxin was identified in previous studies but not examined in terms of possessing emetic activity for primates. rSES induced emetic reactions in 2 of 4 monkeys at a dose of 100 μg/kg within 5 hours of intragastric administration. In one monkey, rSET induced a delayed reaction (24 hours post-administration) at a dose of 100 μg/kg and in the other one, reaction occurred 5 days post-administration. Also, we investigated a mechanism of SEA-induced emesis using a small emetic animal model, House musk shrew (Suncus murinus). SEA induces serotonin (5-hydroxytryptamine, 5-HT) release in intestine, and that the 5-HT3 receptor on vagal afferent neurons are essential for SEA induced emesis. Less
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