Research on the Regulation of Virulence by Cell-Cell Signaling in Pathogenic Bacteria

我们研究了产气荚膜梭菌细胞-细胞信号功能编码基因vapABC的氨基酸序列，并试图鉴定直接参与信号物质产生的基因。我们发现，vapB的信号肽区氨基酸序列与其他细菌的肽信息素非常相似。虽然根据VapB信号区序列合成了4 ~ 20个氨基酸的肽，并将合成的肽添加到产气荚膜荚膜菌中，但未检测到任何显著的活性，表明该信号肽可能被修饰过。然而，由于C.peifringens的vapABC突变体缺乏产生信号分子的能力，我们假设这些基因参与了信号分子的合成，并正在进一步分析。我们还利用DNA微阵列研究了所有基因组基因在细胞与其他细菌之间的通讯中的全局调控。将大肠杆菌与产气荚膜荚膜杆菌共培养，利用DNA芯片分析了细胞-细胞相互作用对产气荚膜荚膜杆菌基因表达的影响。然而，实验结果是失败的，因为背景信号太高，无法进一步分析。混合培养中可能存在一些抑制物质，使实验背景高。现在我们正在努力改善实验条件，并将分析细胞间的相互作用。利用人血清和马血清，探讨影响产气荚膜原菌基因表达的宿主因素。通过DNA微阵列分析鉴定了许多受人血清影响而不受马血清影响的基因。其中包括唾液酸酶等毒力基因，提示人血清中的某些因子可能调控产气荚膜荚膜原菌的毒力表达。

We investigated the amino acid sequence of the vapABC genes encoding a cell-cell signaling function of Clostridium perfringens, and tried to identify the gene directly involved in the production of signal substance. We found that the amino acid sequence in the signal peptide region of vapB was quite similar to those of the peptide pheromones of other bacteria. Although peptides of 4 to 20 amino acids were synthesized based on the sequence of the VapB signal region and the synthetic peptides were added to the cultures of C.perfringens, any significant activities were not detected, suggesting that the signaling peptide might be modified. However, since the vapABC mutant of C.peifringens lacked the ability to produce the signaling molecule, we assume that the genes are involved in the synthesis of the signal molecule and are analyzing further.We also investigated the global regulation of all genomic genes by a cell-cell communication with other bacteria by using DNA microarrays. Escherichia coli was co-cultured with C.perfringens and the effect of the cell-cell interaction on the expression of the genes in C.perfringens was analyzed on DNA microarays. However, the experiments were turned out to fail because the background signal was too high to analyze further. Some inhibitory substances might be present in the mixed culture, which made the background high in the experiments. Now we are trying to improve the experimental condition and are going to analyze the cell-cell interactions.We tried to investigate host factors that influence the expression of C.perfringens genes by using human and horse serum. Many genes that influenced by the addition of human serum but not by horse serum were identified by DNA microarray analysis. Among these genes, virulence genes such as sialidase were included, suggesting some factors in human serum might regulate the expression of virulence in C.perfringens.