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Application of molecular biological technologies to epidemiology of new staphylococcal enterotoxins

分子生物学技术在新型葡萄球菌肠毒素流行病学中的应用

基本信息

批准号：
12660281
负责人：
SHINAGAWA Kunihiro
金额：
$ 2.37万
依托单位：
Iwate University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660281/
关键词：
S. aureus Staphylococcal enterotoxin recombinant DNA emesis diagnosis Staphylococcus aureus protein expression Suncus murinus

项目摘要

In recent years, many new staphylococcal enterotoxins (SEs) have been reported. To Investigate pathobiology and epidemiology of these newly identified SEs, we had undertaken studies shown in below.1. To investigate the distribution of staphylococcal enterotoxin A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from food poisoning outbreaks, from healthy humans, from bovine mastitis, and from bovine raw milk were analyzed by multiplex PCR. One hundred and thirteen (77.4%) S. aureus isolates were diagnosed as positive for one or more se genes. The se genotype was classified into 14 genotypes. The seg, seh and sei genes were detected in S. aureus isolates obtained from all of the sources, seg and sei coexisted in the same S. aureus. The newly developed sandwich ELISA showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most S. aureus isolates harboring seg and about 60% of the isolates ha … More rboring sei did not produce a detectable level of SEG or SEI, while RT-PCR analysis proved that the mRNA of SEG and SEI was transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning (published in Journal of Clinical Microbiology).2. The emetic responses induced by staphylococcal enterotoxin (SE) A to SEE and SEG to SEI in house musk shrew (Suncus murinus) were investigated. SEA, SEE and SEI showed higher emetic activity in house musk shrew. SEB, SEC, SED, SEG and SEH also induced emetic responses in this animal model but required relatively high dose. House musk shrew appears to be a valuable model for studying of the mechanisms of emetic reaction caused by staphylococcal enterotoxins (published in Infection and Immunity).3. We identified and characterized a novel staphylococcal superantigen, named SER, which is phylogenetically related to staphylococcal enterotoxin (SE). The SER encoding gene, ser, was cloned from Staphylococcus aureus isolate that was involved in an SE-unidentified food poisoning outbreak that occurred in Japan. This isolate harbored another se gene, sej. Both ser and sej genes were located on the same 2.8 kbp EcoRI fragment and these open reading frames are transcribed in opposite directions. Nucleotides sequencing analysis revealed that SER was most closely related to SEG, with 65.9% (nucleotide sequence) and 56.4% (amino acid sequence) identity. Both ser and sej transcriptions were confirmed by Northern hybridization. The ser gene product, SER, was successfully expressed as recombinant protein in an E. coli expression system, and a monospecific polyclonal antibody was prepared against recombinant SER. Using this antibody, we were able to detect SER production by immunoblot in ser harboring S. aureus strains. Recombinant SER protein expressed by E. coli showed significant superantigenic activities. Moreover, ser was seen to be encoded by at least two types of plasmids. In particular, one kind of plasmid encoding the ser gene has been known as sed- and sej-encoding pIB485-related plasmid (submitted). Less

近年来，许多新的葡萄球菌肠毒素（SE）被报道。为了研究这些新发现的SE的病理生物学和流行病学，我们进行了以下研究。为了研究金黄色葡萄球菌肠毒素A至I（SEA至SEI）基因（SEA至sei）在金黄色葡萄球菌中的分布，采用多重PCR方法分析了从日本食物中毒爆发、健康人、牛乳腺炎和牛原料奶中获得的146株金黄色葡萄球菌。113个（77.4%）S.金黄色葡萄球菌分离株被诊断为一种或多种SE基因阳性。Se基因型分为14种。在S.所有来源的金黄色葡萄球菌分离株，seg和sei共存于同一株金黄色葡萄球菌中。金黄色。新建立的双抗体夹心ELISA法显示，大多数携带seh的S。金黄色葡萄球菌分离株能够产生显著量的SEH。然而，大多数S。携带SEG的金黄色葡萄球菌分离株和大约60%的分离株HA ...更多信息转染sei的S.携带SEG和SEI基因的金黄色葡萄球菌菌株。这些结果表明定量评估食品中SEG和SEI的产生以澄清这些新SE与食物中毒之间的关系的重要性（发表在Journal of Clinical Microbiology）.研究了金黄色葡萄球菌肠毒素A（SE A）和SEG对SE I的催吐反应。SEA、SEE和SEI对家麝鼩有较强的催吐活性。SEB、SEC、SED、SEG和SEH也在该动物模型中诱导呕吐反应，但需要相对较高的剂量。家麝鼩似乎是研究葡萄球菌肠毒素引起呕吐反应机制的有价值的模型（发表在Infection and Immunity）.我们鉴定并鉴定了一种新的葡萄球菌超抗原，命名为SER，它与葡萄球菌肠毒素（SE）在遗传学上相关。SER编码基因，ser，是从金黄色葡萄球菌分离株，涉及在日本发生的SE不明的食物中毒爆发。该分离株携带另一个se基因sej。ser和sej基因位于同一个2.8 kbp的EcoRI片段上，这些开放阅读框以相反的方向转录。核苷酸序列分析表明，SER与SEG的同源性最高，分别为65.9%（核苷酸序列）和56.4%（氨基酸序列）。通过北方杂交证实了ser和sej transcription。将ser基因产物SER在大肠杆菌中成功表达。大肠杆菌表达系统，并制备了重组SER的单特异性多克隆抗体，利用该抗体，我们能够用免疫印迹法检测含SER的S.金黄色葡萄球菌重组SER蛋白在E. coli中具有明显的超抗原活性。此外，ser被认为是由至少两种类型的质粒编码。特别是，已知一种编码ser基因的质粒为编码sed和sej的pIB 485相关质粒（已提交）。少