Analysis of Maturation pathway protein toxin of Enteric Bacteria and Studies of Prevention of Diarrhea

本研究对产肠毒素的肠毒素和产肠毒素大肠杆菌的肠毒素成熟途径、产气单胞菌的蛋白水解酶和耐热肠毒素进行了研究。气单胞菌的致泻毒素对红细胞有很强的溶解作用，故又称溶血素。温和气单胞菌和嗜水气单胞菌的培养滤液具有溶血活性，因为溶血素是在溶液中产生的。然而，鼠疫杆菌的滤液没有表现出来，然而，鼠疫杆菌引起了人类的腹泻。我们对豚鼠腹泻性毒素的产生进行了检测。已有研究表明，鼠疫杆菌可合成溶血素，并将其释放到细胞外，但溶血素被其产生的蛋白酶分解。因此，豚鼠培养滤液不具有溶血活性。在肠腔和琼脂介质中，这些由豚鼠产生的溶血素可以结合适当的细胞并在这些溶血素被蛋白酶破坏之前表达出活性。本研究项目的另一个发现是，温和气单胞菌的丝氨酸蛋白酶和金属蛋白酶的产生受到盐(氯化钠)的抑制。在含有2%以上氯化钠的介质中，这些酶的产生受到显著抑制。对抑制机制的研究表明，丝氨酸蛋白酶和金属酶的产生抑制分别来源于对成熟途径和转录阶段的抑制。在对大肠杆菌的研究中，研究了与耐热肠毒素(ST)跨外膜转运有关的TolC的结构。为了进行检测，我们构建了由大肠杆菌TolC和副溶血性弧菌TolC组成的嵌合TolC。结果表明，E.ColiTolC第198-214位区域在ST的转运过程中起着重要作用。

The studies on maturation pathway of enterotoxin and protease of Aeromonas and heat-stable enterotoxin of enterotoxigenic Escherichia coil were performed in this study. The diarrheagenic toxin of Aeromonas has rapacity to lyse erythrocytes, therefore, another name the toxin is hemolysin. The culture filtrates of A. sobria and A. hydrophila exhibit hemolytic activity, because the hemolysin is produced into the solution. However, the filtrate of A. caviae does not exhibit However, A. caviae caused diarrhea in humans. We examined the production of diarrheagenic toxin of A. caviae. It has been shown that A. caviae synthesis the hemolysin and release it into outside of the cells, however, the hemolysin is broken by the proteases produced by A. caviae. Therefore, the culture filtrate of A. caviae does not exhibit hemolytic activity. In intestinal lumen and agar medium, these hemolysins produced by A. caviae can bind the appropriate cells and express the activity before these hemolysins are broken by proteases. Another finding of this research project is that production of serineprotease and metallo protease of A. sobria is inhibited by salt (sodium chloride) in the medium. hi the medium containing sodium chloride at the ratio of more than 2%, the production of these proteases is significantly repressed. The studies on the mechanism of the repression showed that the repressions of production of serineprotease and metallo pro ease are derived from inhibitions of maturation pathway and of transcriptional stage of each toxin, respectively. In the study about Escherichia coil, the structure of TolC which is involved in the translocation of ST(heat-stable enterotoxin) across the outer membrane was examined. To examined, we constructed the chimeric TolC which is consisted of E. coli TolC and Vibrio parahaemolyticus TolC. The result shows that the region covering 198th -214th of E. coli TolC plays an important role in the transport of ST.