Correlation between infection mechanisms and virulence plasmid of Salmonella choleraesuis

1. 猪霍乱沙门氏菌毒力质粒pKDSC50与小鼠毒力的相关性：将携带毒力质粒pKDSC50（亲本株和再引入株）的猪霍乱沙门氏菌菌株和治愈株腹腔接种ICR小鼠，对小鼠进行14 d的病理检查。亲本菌株和重新引入的菌株引起小鼠菌血症，接种的细菌从脾脏和肝脏中恢复。腹膜液、肠系膜、内脏器官各组织均有明显病理改变，并持续到第14天。相比之下，感染质粒治愈菌株的小鼠未出现菌血症，仅在腹膜液、肠系膜和内脏发生轻微和短暂的病理改变。比较了含pkdsc50的霍乱链球菌与固化菌株的细胞表面组分差异：亲本菌株、再引入菌株和固化菌株的脂多糖、外膜蛋白和磷脂。两者之间无显著差异，说明pKDSC50没有介导细菌细胞表面可检测的变化。转座子插入失活引起小鼠菌血症能力的区域定位：在病理研究结果的基础上建立小鼠菌血症试验，用于筛选转座子tnl插入失活构建的100株突变株。将小鼠菌血症试验阴性菌株的质粒DNA转化到治愈菌株中，再次检测转化菌株的小鼠菌血症能力。9株菌株第二次检测阴性，其质粒上的Tnl定位在pKDSC50的6.2 kb区域。这个地区被指定为mba地区。pKDSC50 mba区域的遗传研究：在大肠杆菌中克隆了mba区域的DNA片段，并利用大肠杆菌微细胞系统或异丙基硫半乳糖诱导的lac-启动子系统对该区域编码的蛋白进行了鉴定。检测到表观分子量分别为29k、70k、32k和32k的4种蛋白，通过核苷酸序列分析在mba区发现了与这4种蛋白对应的开放阅读框。近年来，其他研究小组报道了鼠伤寒沙门氏菌毒力质粒中编码的类似蛋白，表明我们发现的蛋白在沙门氏菌毒力质粒中是常见的，并且在沙门氏菌的发病机制中起重要作用。少

1. Correlation between virulence plasmid of Salmonella choleraesuis, pKDSC50, and its mouse virulence: S. choleraesuis strains harboring virulence plasmid, pKDSC50 (parent and re-introduced strains), and cured strains were inoculated intraperitoneally into ICR mice, and the mice were examined pathologically for 14 days. Parent and re-introduced strains caused bacteremia in mice, and inoculated bacteria were recovered from spleen and liver. Obvious pathological changes were observed at individual tissues of peritoneal fluid, mesenterium, and visceral organs, and those changes persisted to day 14. In contrast, mice infected with plasmid-cured strain showed no bacteremia, and had only mild and transient pathological changes at peritoneal fluid, mesenterium, and viscera.2. Difference of cell surface components between pKDSC50-harbouring and -cured strains of S. choleraesuis : Lipopolysaccharide, outer membrane proteins, and phospholipids of parent, re-introduced, and cured strains were che … More mically and biochemically compared. There was no significant difference between them, indicating that pKDSC50 did not mediate the detectable change of bacterial cell surface.3. Mapping of the region responsible for the ability to cause mouse bacteremia by transposoninsertional inactivation: Mouse bacteremia test was established in the base of the results of pathological study, and used for screening 100 mutant strains constructed by transposon Tnl-insertional inactivation. Plasmid DNA of mouse bacteremia test-negative strains were introduced to cured strain by transformation, and the transformants were again tested for mouse bacteremia ability. Nine strains were negative for the second test, and Tnl on their plasmids were localized in 6.2 kb region of pKDSC50. This region was designated mba region.4. Genetical study on mba region of pKDSC50: DNA fragments of mba region were cloned in E. coli, and proteins encoded in this region were identified by E. coli minicell system, or by lac-promoter system with induction of isopropylthiogalactoside. Four proteins with apparent molecular weights of 29k, 70k, 32k, and 32k were detected, and open reading frames corresponding to these four proteins were found in mba region by nucleotide sequential analysis. Other research groups has recently reported similar proteins encoded in virulence plasmid of S. typhimurium, suggesting that the proteins we found were common among virulence plasmid-harboring Salmonella, and playing important role in pathogenesis of Salmonella. Less