单增李斯特菌（EGDe）sigB基因缺失株的构建及其生物特性的初步鉴定

Construction of Attenuated Listeria monocytogenes Strain EGDe-sigB and Preliminary Identification of Biological Activity of sigB

摘要:

单核细胞增生李斯特氏菌（Listeria monocytogenes，Lm）是一种人畜共患的食源性致病菌，在感染过程中可以穿越人体肠道屏障、胎盘屏障及血脑屏障，进而引发肠胃炎、脑膜炎及孕妇流产等疾病。Sigma B (sigB)基因编码产生的sigmaB因子是许多革兰氏阳性菌对环境胁迫产生应答反应的主要调控因子，并且直接或间接的调控Lm中相关重要毒力基因如prfA、inlA等的表达。本文通过同源重组技术将Lm野生菌株EGDe的sigB基因敲除，并且对缺失菌株的生长曲线，inlA、inlB、prfA等13种Lm的毒力基因表达水平及对肠道上皮细胞Caco-2的侵袭进行了研究。实验表明，EGDe-Δ sigB基因缺失菌株生长速度与EGDe基本一致；sigB基因的缺失造成inlA和inlB基因表达水平的大幅度下调，但actA和plcA等毒力基因却上调4~5倍，说明sigB基因对于单核增生性李斯特菌的一些毒力基因表达影响比较大；Caco-2细胞的侵袭实验显示EGDe-Δ sigB基因的缺失对Caco-2细胞侵袭能力的下降。

Abstract:

Listeria monocytogenes is a foodborne pathogen infectious to both humans and animals. L. monocytogenes can cross the human intestinal barrier, placental barrier, and blood-brain barrier, thereby causing gastroenteritis, meningitis, miscarriage, and other diseases. Sigma B factor, encoded by the gene sigB, is an important regulatory factor for the environmental stress response in many Gram-positive bacteria, and directly and indirectly controls the expression of important virulence genes, such as prfA and inlA. The sigB gene of L. monocytogenes strain EGDe was knocked out by homologous recombination, and the resulting deletion strain (EGDe-ΔsigB) was used to study the effect of sigB deletion on growth, expression of 13 virulence genes (including inlA, inlB, and prfA), and invasion of Caco-2 intestinal epithelial cells. The results showed that the growth rate of the EGDe-ΔsigB strain was equivalent to that of the wild-type EGDe strain. Deletion of sigB caused a significant reduction in the expression levels of inlA and inlB, but a significant (four- to five-fold) increase in the expression levels of the virulence genes actA and plcA, demonstrating that sigB has a relatively large influence on the expression of some virulence genes in L. monocytogenes. Furthermore, invasion assays indicated that deletion of sigB caused a decline in the ability of L. monocytogenes strain EDGe to invade Caco-2 cells.