Comparative genomics of staphylococcal enterotoxin gene family

近年来，许多新的葡萄球菌肠毒素（SEs）被报道。已知某些SE基因与可移动的遗传元件如致病性岛、噬菌体和质粒有关。这些事实表明，超抗原毒素基因在葡萄球菌菌株之间水平转移。有一种可能性是，这些可移动的遗传元素在金黄色葡萄球菌作为病原体的进化中发挥了重要作用。为了调查这些新发现的Ses的病理生物学和流行病学，我们进行了如下所示的研究。为了检测和鉴定编码致病性岛的未知SE基因，我们开发了一种新的方法——致病性岛扫描。基于几种金黄色葡萄球菌的全基因组序列，我们设计了PCR引物来扩增整个致病性岛屿区域。扩增子进行限制性片段长度多态性（RFLP）分析。利用致病性岛扫描/RFLP技术，我们发现seb相关致病性岛至少可分为5类。此外，我们还利用霰弹枪测序策略确定了selj/selr编码质粒pF5的完整核苷酸序列。该质粒除了编码selj和selr外，还编码了两个新的肠毒素基因ses和set。此外，我们还开发了几种诊断方法，包括免疫PCR敏感检测SE，培养/PCR敏感检测金黄色葡萄球菌，多重PCR综合检测SE/SE1基因。我们鉴定了两种新型葡萄球菌肠毒素SES和SET。重组(r)SES在MHC II类^+抗原呈递细胞（APCs）存在下，以T细胞受体（TCR） v β9特异性刺激人T细胞。在MHC II类APC存在时，rSET也刺激T细胞，尽管在反应性T细胞中未发现其Vβ偏斜。随后，我们检测了SES和SET的催吐活性。我们还研究了SEIR来确定灵长类动物的呕吐活动。这种毒素在以前的研究中被发现，但没有在灵长类动物的呕吐活动方面进行检查。给药剂量为100 μg/kg时，4只猴子中有2只在5小时内出现呕吐反应。在给药剂量为100 μg/kg时，一只猴子出现延迟反应（给药后24小时），另一只猴子在给药后5天出现反应。此外，我们还利用小型呕吐动物家鼠鼩（Suncus murinus）研究了sea诱导的呕吐机制。SEA诱导肠内5-羟色胺（5-羟色胺，5-HT）释放，迷走神经传入神经元上的5-HT3受体在SEA诱导的呕吐中起重要作用。少

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 … More 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