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

本研究通过对产气荚膜梭菌的细胞-细胞信号转导功能基因VapABC的氨基酸序列进行分析，试图找出与信号物质的产生直接相关的基因。我们发现，VAPB信号肽区的氨基酸序列与其他细菌的多肽信息素非常相似。虽然根据VAPB信号区的序列合成了4~20个氨基酸的多肽，并将合成的多肽加入产气荚膜梭菌的培养中，但没有检测到任何显著的活性，提示信号肽可能被修饰。然而，由于Peifringens的VapABC突变体缺乏产生信号分子的能力，我们推测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通过与其他细菌的细胞间通讯而进行的全局调控。将大肠杆菌与产气荚膜梭菌共培养，利用DNA微阵列技术分析细胞间相互作用对产气荚膜梭菌基因表达的影响。然而，实验被证明是失败的，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，使实验中的背景较高。现在我们正在努力改善实验条件，分析细胞间的相互作用。我们试图利用人和马的血清来研究宿主因素对产气荚膜梭菌基因表达的影响。通过DNA微阵列分析，发现了许多受人血清影响而不受马血清影响的基因。在这些基因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节产气荚膜梭菌毒力的表达。

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.