关键词: 单核细胞性李斯特菌, 基因敲除, 生长能力, 细胞侵袭, 生物被膜

Abstract: In this study, the actA and inlB double gene deletion mutant (EGDe ΔactAΔinlB) of Listeria monocytogenes wild-type (WT) strain EGDe was used as the parent to delete the dal gene by homologous recombination technology, and the biological characteristics of the dal-deficient mutant such as growth capacity, virulence gene expression, cell invasion and biofilm formation were further studied. Growth curves showed that the concentration of the new mutant strain was significantly lower than that of EGDe ΔactAΔinlB after 6 h of shaking culture at 37 ℃ (P < 0.001), but there was no difference in the growth rates of the parental and the mutant strains when D-alanine was added to the medium. Quantitative real-time-PCR showed that the sigB gene expression level of the mutant strain was changed most significantly (P < 0.01) and down-regulated by 90% compared with EGDe ΔactAΔinlB. The biofilm formation of the mutant strain increased compared with EGDe ΔactAΔinlB, but this difference did not exist when D-alanine was added to the medium for the mutant. There was no significant difference in Caco-2 cells invasion ability compared with EGDe ΔactAΔinlB. The results indicated that the dal gene played an important regulatory role in the growth and biofilm formation of bacteria and did not affect the ability of cell invasion. The construction of this deletion strain can provide a tool for further study of the function of the dal gene.

Key words: Listeria monocytogenes, gene knockout, growth capacity, biofilm, cell invasion