Research on pathogenic gienes and development of vaccine of Vibrio valunificus, a novel causative agent of foodborne infection

创伤弧菌是食源性疾病和伤口感染的重要致病菌。这种病原体在免疫功能低下的宿主接触带有这种有机体的受污染的海水或海鲜后，会迅速引起致命的败血症。死亡率超过50%。由于进展迅速，死亡率高，疫苗接种被认为是控制高危人群创伤弧菌感染的有效途径。我们应用特征标记诱变（STM）技术从临床分离的创伤弧菌毒力菌株OPU1中产生减毒突变体，该菌株从一名患者身上分离出来。选取12个插入突变体，采用最小致死剂量法测定其对小鼠的毒力。几个突变株的毒力低于亲本毒株。测定了这些突变体转座子插入位点侧翼的DNA序列，并在日本DNA数据银行进行了同源性检索。所插入的转座子基因为IMP脱氢酶、udp - n-乙酰葡萄糖胺-2- epimase、天冬氨酸激酶和一种保守的假设蛋白。这表明这些基因可能与创伤弧菌的毒力有关。其中一种突变体被用于小鼠的实验性疫苗接种。免疫小鼠用毒力强的亲本毒株攻毒。未免疫小鼠在36小时内全部死亡，而免疫小鼠几乎全部存活。在本实验中，我们发现该减毒疫苗具有作为活疫苗候选疫苗的潜力。

Vibrio vulnificus is an important pathogens of foodborn diseases and wound infection. This pathogen causes fatal septicemia rapidly in immunocompromised hosts after they contact with contaminated seawater or seafood with this organism. The mortality rate is more than 50%. For the rapid progress and the high mortality,vaccination is considered an effective way to control infection with V. vulnificus in a high risk population. We applied Signature-Tagged Mutagenesis (STM) to generate attenuated mutants from a clinically-isolated virulent V. vulnificus strain, OPU1 which was isolated from a patient. Twelve insertion mutants were selected and the virulence of these mutants were determined by the minimal lethal dose (MLD) to mice. Several mutant strains showed lower virulence compare to the parent virulent strain. The DNA sequences flanking to transposon insertion sites in these mutants were determined and searched for homologies at DNA Date Bank of Japan. The transposons inserted genes were those for IMP dehydrogenase, UDP-N-acetylgulcosamin-2-epimerase, aspartokinase, and a conserved hypothetical protein. This suggests that these genes may be involved in V. vulnificusvirulence. One of the mutants was administrated for experimental vaccination to mice. The immunized mice were challenged with the virulent parent strain. All unimmunized mice were killed within 36 hr, but almost all immunized mice survived. In this experiment, we found that the attenuated have a potentiality as live vaccine candidate.