关键词: 鼠伤寒沙门菌, c-di-GMP, STM2503, 生物被膜, 运动性

Abstract:

【Objective】S. Typhimurium is an important zoonotic pathogen, and its strong pressure adaptability brings great challenges to its prevention and control. In this study, the mechanism related to c-di-GMP pathway gene STM2503 regulating the biofilm formation, and thereby affects environmental stress capacity of S. Typhimurium, were investigated, so as to excavate the key factors regulating the stress adaptation of S. Typhimurium, and provide a theoretical basis for the formulation of new prevention and control strategies.【Method】The STM2503 gene deletion strain was constructed by λ-Red homologous recombination, and plasmid PBAD was used to express STM2503 to construct the gene complementation strain. Then, the effect of STM2503 on the intracellular c-di-GMP level of bacteria was revealed by detecting the signaling molecule c-di-GMP content in strains with different STM2503 expression levels. Next, the effect of STM2503 on bacterial motility and biofilm formation ability were detected by bacterial motility detection and crystal violet staining experiments, furthermore, the real-time fluorescence quantitative PCR was employed to elucidate its regulatory mechanism on bacterial motility and biofilm formation at the genetic level. Finally, the effect of STM2503 on the stress adaptability of S. Typhimurium was investigated through antibiotic treatment, oxidative stress, and disinfectant stress tests.【Result】Compared with the wild strain (WT269), the deletion of STM2503 increased the intracellular c-di-GMP content of the strain by 37.51%, and did not affect the normal growth of the strain. In addition, the deletion of STM2503 increased the expression level of extracellular matrix synthesis genes, and increased the content of extracellular polysaccharide, extracellular DNA and extracellular protein by 10.30% (P<0.01), 33.59% (P<0.001) and 27.60% (P<0.01), respectively, which ultimately led to a significant enhancement of biofilm formation ability by 1.63-fold (P<0.01). Moreover, 269ΔSTM2503 reduced the motile diameter by 17.22% (P<0.01) compared with the wild strain (WT269) at 6 h via decreasing the expression of flagella synthesis related genes fliA and flhC. These changes eventually led to a 1-3 fold reduction in the sensitivity of the 269ΔSTM2503 strain to β-lactam antibiotics, such as cefotaxime, cefepime, and amoxicillin, along with showed stronger adaptability under oxygen stress and SDS disinfectant stress. 【Conclusion】 In conclusion, STM2503 was involved in the degradation of the signaling molecule c-di-GMP and reduced the biofilm formation ability of S. Typhimurium by inhibiting the synthesis of extracellular substances and enhanced the motility of strains by upregulating the expression of flagellar synthesis genes, thereby reducing the drug resistance and environmental stress adaptability of S. Typhimurium. This study provided a theoretical basis for the excavation of the key targets for the prevention and control of S. Typhimurium.

Key words: S. Typhimurium, C-di-GMP, STM2503, biofilm, motility