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

摘要/Abstract

摘要： 【目的】构建糖基翻转酶gtcA基因缺失株，并阐明其对单增李斯特致病性的影响。【方法】利用同源重组技术构建gtcA基因缺失株；进一步分析亲本株EGDe-prfA*、缺失株ΔgtcA和回补株CΔgtcA的生长能力；通过Western blotting分析gtcA基因缺失对InlA和InlB在细菌表面锚定的影响；通过DF1细胞黏附侵袭试验、RAW264.7细胞吞噬增殖试验和鸡胚毒力试验评估gtcA基因缺失对单增李斯特菌细胞侵染能力及对鸡胚致病性的影响。【结果】生长曲线显示，gtcA基因缺失并不影响单增李斯特菌在BHI培养基中的生长能力。Western blotting结果显示，内化素InlB在ΔgtcA表面的锚定量极显著低于EGDe-prfA*和CΔgtcA（P<0.01），但其表面InlA的锚定量与EGDe-prfA*并无显著差异（P>0.05）。细胞黏附侵袭试验结果显示，ΔgtcA对成纤维细胞DF1的平均黏附率和侵袭率显著低于EGDe-prfA*和CΔgtcA（P<0.05）。吞噬试验结果显示，巨噬细胞RAW264.7对ΔgtcA的平均吞噬率显著低于EGDe-prfA*和CΔgtcA（P<0.05），但ΔgtcA与EGDe-prfA*、CΔgtcA在RAW264.7中的3 h内增殖倍数并无显著差异（P>0.05）。鸡胚毒力试验结果显示，ΔgtcA感染鸡胚肝脏和脾脏中的平均细菌载量分别为6.86×10³和3.69×10² CFU，极显著低于EGDe-prfA*和CΔgtcA感染鸡胚（P<0.01）；同时，ΔgtcA感染鸡胚存活率及存活时间均高于EGDe-prfA*和CΔgtcA感染鸡胚。【结论】糖基转移酶gtcA基因缺失不影响单增李斯特菌1/2a血清型菌株EGDe-prfA*的生长能力，但能显著降低该菌表面InlB的锚定丰度，减弱该菌的细胞侵染能力及对鸡胚的致病性。本研究结果为深入探索糖基翻转酶基因gtcA介导单增李斯特菌致病机制提供参考。

关键词: 单增李斯特菌; 糖基翻转酶; gtcA基因; 缺失; 致病性

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

中图分类号:

S852.61⁺7

郭骞, 张钰, 方小伟, 安妮, 袁梅, 方春. 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[J]. 中国畜牧兽医, 2022, 49(6): 2298-2306.

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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