糖基翻转酶gtcA基因缺失降低单增李斯特菌1/2a血清型菌株致病性的研究

doi:10.16431/j.cnki.1671-7236.2022.06.030

Abstract

Abstract: 【Objective】 This study was aimed to construct the glycosyl-flippase gtcA gene deletion strain, and explore the effects of gtcA gene in the pathogenicity of Listeria monocytogenes.【Method】 The deletion strain ΔgtcA was constructed by homologous recombination, the growth ability of EGDe-prfA*, ΔgtcA and CΔgtcA was compared.The effects of gtcA gene deletion on the anchoring of InlA and InlB on bacterial surface was detected by Western blotting.The roles of gtcA gene in pathogenicity was validated by the adhesion and invasion assay on DF1 cells, the phagocytosis and multiplication assay in macrophages RAW264.7, and virulence assay conducted on chicken embryo.【Result】 The growth curve showed that gtcA gene deletion did not affect the growth ability of Listeria monocytogenes in BHI medium.Western blotting results showed that the anchor quantity of InlB in ΔgtcA surface was extremely significantly lower than that of EGDe-prfA* and CΔgtcA (P<0.01), but the anchor quantity of InlA in ΔgtcA surface was not significantly lower than that of EGDe-prfA* (P>0.05).The results of cell adhesion and invasion test showed that the average adhesion rate and invasion rate of ΔgtcA were significantly lower than that of EGDe-prfA* and CΔgtcA (P<0.05).The phagocytosis test results showed that the average phagocytosis rate of ΔgtcA by macrophage RAW264.7 was significantly lower than that of EGDe-prfA* and CΔgtcA (P<0.05), but there was no significant difference of proliferation multiple within 3 h in macrophage RAW264.7 between ΔgtcA and EGDe-prfA* or CΔgtcA (P>0.05).Virulence assay results showed that the average bacteria load of liver and spleen in ΔgtcA infected chicken embryo were 6.86×10³ and 3.69×10² CFU, respectively, which were extremely significantly lower than that of EGDe-prfA* and CΔgtcA infected chicken embryos (P<0.01).Moreover, the survival rate and survival time of ΔgtcA infected chicken embryo were higher than that of EGDe-prfA* and CΔgtcA infected chicken embryos.【Conclusion】 gtcA gene deletion did not affect the growth ability of EGDe-prfA*, while decreased the surface anchoring of InlB, infection ability and pathogenicity of EGDe-prfA*.The results provided a reference for further exploring the mechanisms of gtcA mediates pathogenicity of Listeria monocytogenes.

Key words: Listeria monocytogenes; glycosyl-flippase; gtcA gene; deletion; pathogenicity

CLC Number:

S852.61⁺7

GUO Qian, ZHANG Yu, FANG Xiaowei, AN Ni, YUAN Mei, FANG Chun. Study on Glycosyl-flippase gtcA Gene Deletion Reduces the Pathogenicity of Listeria monocytogenes Serotype 1/2a[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2298-2306.

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Study on Glycosyl-flippase gtcA Gene Deletion Reduces the Pathogenicity of Listeria monocytogenes Serotype 1/2a

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