The significance of Chromosomal Virulence Genes of Shigella flexneri in the Pathogenesis of Bacillary Dysentery

志贺氏杆菌导致痢疾症状的基本步骤是侵入结肠上皮细胞，然后是细胞内细菌增殖和入侵细菌向邻近细胞扩散。志贺氏菌毒力的遗传决定因素位于230 kb的质粒和染色体上。许多实验室的研究人员对质粒毒力基因进行了广泛的分子研究。我们在之前的项目中也进行了这样的研究，结果表明，质粒编码的毒力相关功能涉及细胞内入侵、毒力蛋白分泌、细胞内和细胞间扩散以及这些质粒编码的毒力基因表达的调控。本项目是这些研究的延伸，旨在阐明染色体毒力基因与质粒基因在细菌性痢疾发病机制中的作用。结果如下：(1)采用随机转座子插入诱变法分离出无毒的转座子插入突变体。(2)分析了这些突变体的毒力相关表型。(3)构建了该染色体的NotI限制性切割图谱，并在其上分配了上述突变体。(4)通过这些方法鉴定的一些基因座进一步进行了遗传分析和细胞生物学技术。这些毒力相关区域被发现参与(I) LPS生物合成，（II）细胞内增殖，（III）细胞间扩散，或（IV）大质粒上毒力基因表达的调控。对于被划分为（III）或（IV）类的区域，已经进行了详细的分析，结果已经作为文章或评论发表在国际期刊上。

The essential steps leading to dysenteric symptomes by the bacterium Shigella is invasion of colonic epithelial cells, followed by intracellular bacterial multiplication and spread of invading bacteria to adjacent cells. The genetic determinants for the virulence of shigellae are located on the large 230 kb plasmid as well as on the chromosome. Extensive molecular studies have been performed on the plasmid virulence genes by the investigators from many laboratories. We also engaged in such studies in the previous project which resulted in showing that the plasmid encodes virulence-associated functions involved in intracellular invasion, secretion of virulence proteins, intra-and intercellular spreading and regulation of the expression of these plasmid-coded virulence genes. The present project was an extention of these studies aiming to clarify the significance of chromosomal virulence genes in collaboration with the plasmid genes in the pathogenesis of bacillary dysentery. The following are the summary of the results obtained:(1) Avirulent transposon insertion mutants were isolated by random transposon insertion mutagenesis. (2) The virulence-associated phenotypes of these mutants were analysed. (3) A NotI restriction cleavage map of the chromosome was constructed and the above-described mutants were assigned on it. (4) Some of the loci identified by these methods were analysed further genetically and by cellular biological techiques. These virulence-associated regions were found to be involved in either (I) LPS biosynthesis, (II) intracellular multiplication, (III) intercellular spreading, or (IV) regulation of the expression of the virulence genes on the large plasmid. For the regions classified into category (III) or (IV), a detailed analysis was made and the results have already been published as articles or reviews in international journals.