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脂磷壁酸合成相关基因dltD对高致龋力变异链球菌株耐酸能力的影响

杜景云 吴敏婧 李艺君 黄珊 江山 陈帅 黄晓晶

杜景云, 吴敏婧, 李艺君, 等. 脂磷壁酸合成相关基因dltD对高致龋力变异链球菌株耐酸能力的影响[J]. koko体育app 学报(医学版), 2022, 53(2): 235-241. doi: 10.12182/20220360102
引用本文: 杜景云, 吴敏婧, 李艺君, 等. 脂磷壁酸合成相关基因dltD对高致龋力变异链球菌株耐酸能力的影响[J]. koko体育app 学报(医学版), 2022, 53(2): 235-241. doi:
DU Jing-yun, WU Min-jing, LI Yi-jun, et al. Effect of Lipoteichoic Acid Synthesis-Related Gene dltD on Acid Tolerance of Highly Cariogenic Strains of Streptococcus mutans[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2022, 53(2): 235-241. doi: 10.12182/20220360102
Citation: DU Jing-yun, WU Min-jing, LI Yi-jun, et al. Effect of Lipoteichoic Acid Synthesis-Related Gene dltD on Acid Tolerance of Highly Cariogenic Strains of Streptococcus mutans[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2022, 53(2): 235-241. doi:

脂磷壁酸合成相关基因dltD对高致龋力变异链球菌株耐酸能力的影响

doi: 
基金项目: 国家自然科学基金(No. 81600861)和闽江学者科研启动基金(No. 2018-KQMJ-01)资助
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    通讯作者:

    E-mail:xiaojinghuang@fjmu.edu.cn

Effect of Lipoteichoic Acid Synthesis-Related Gene dltD on Acid Tolerance of Highly Cariogenic Strains of Streptococcus mutans

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  • 摘要:   目的  通过构建变异链球菌593(Streptococcus mutans 593, SM593)dltD基因缺失株,探究dltDSM593耐酸能力中的作用和可能机制,为龋病的生态防治提供理论依据。  方法  ①同源重组构建SM593 dltD基因缺失株SM593-ΔdltD;②采用全自动生长曲线分析仪绘制在不同pH培养条件下SM593和SM593-ΔdltD的生长曲线,比较二者耐酸能力;通过SM593和SM593-ΔdltD不同时间点的菌落计数(colony forming unit, CFU)计算其生存率并比较二者耐酸反应(acid tolerance response, ATR)能力;③通过不同pH条件下的糖酵解能力检测、质子通透性检测、质子移位膜腺苷三磷酸酶(H+-ATPase)活性检测初步探究dltD基因缺失影响耐酸的可能机制。  结果  ①PCR及测序结果显示SM593 dltD基因缺失株构建成功;②随着培养基pH逐渐降低,SM593-ΔdltD生长减缓,当培养基pH=5.0时无法生长,与 SM593相比耐酸能力下降;SM593-ΔdltDSM593相比ATR能力下降;③不同pH条件下,SM593-ΔdltDSM593相比,糖酵解能力无明显差异,质子通透性增加(P<0.05),H+-ATPase活性下降(P<0.05)。  结论  dltD基因缺失株较原始株相比耐酸能力明显下降,可能是由于dltD基因缺失导致质子通透性显著增强、H+-ATPase活性显著降低,从而使菌株维持胞内pH稳态能力下降所致。
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    图  1  SM593 dltD基因缺失株鉴定结果

    Figure  1.  Identification result of SM593 dltD gene deletion strain

    A: Positive re-cloning screening (containing 1 g/L spectinomycin); Ba: The gel electrophoresis result of the suspected dltD gene deletion strain is primarily screened by PCR; Bb: The gel electrophoresis result of the suspected dltD gene deletion strain is verified by PCR; C: BLAST results of sequencing of suspected dltD gene deletion strains.

    图  2  SM593和SM593-ΔdltD在不同pH的BHI培养基中的生长曲线(n=3)

    Figure  2.  Growth curves of SM593 and SM593-ΔdltD in BHI medium of different pH values (n=3)

    图  3  SM593和SM593-ΔdltD耐酸反应能力比较(n=3)

    Figure  3.  Comparison of acid tolerance response between SM593 and SM593-ΔdltD (n=3)

    图  4  SM593和SM593-ΔdltD在不同pH条件下糖酵解能力比较(n=3)

    Figure  4.  Comparison of glycolysis abilities of SM593 and SM593-ΔdltD at different pH values (n=3)

    图  5  SM593和SM593-ΔdltD在不同pH条件下质子通透性比较(n=3)

    Figure  5.  Comparison of proton permeability of SM593 and SM593-ΔdltD at different pH conditions (n=3)

    *P<0.05, vs. SM593 at the same time.

    图  6  SM593和SM593-ΔdltD在不同pH条件下H+-ATPase活性比较(n=3)

    Figure  6.  Comparison of H+-ATPase activities between SM593 and SM593-ΔdltD under different pH conditions (n=3)

    * P<0.05.

    表  1  本实验所用引物

    Table  1.   Primers used in this experiment

    Primer nameSequenceApplication
    dltD-up-F 5′-CGGATCCCCGTTTTATGCTGTTTATGCCTACT-3′ 5′ fragment for dltD mutation
    dltD-up-R 5′-CCCAAGCTTAAACTGGCCCCAAAATCAACC-3′
    dltD-down-F 5′-CCCATGGGGGCCACTTATACTGGTAATCCT-3′ 3′ fragment for dltD mutation
    dltD-down-R 5′-CCCCCGGGACAGCTCTCACCGTCTTTCA-3′
    dltD-in-F 5′-TGGTTCGTTCCAAAGGGGAC-3′ dltD sequencing confirmation
    dltD-in-R 5′-CGATCCGCTGTACTTCCTGT-3′
    aad9-F 5′-TTGGATCAGGAGTTGAGAGTGGAC-3′ spe sequencing confirmation
    aad9-R 5′-GCCACTGCATTTCCCGCAATATCT-3′
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出版历程
  • 收稿日期:  2022-08-08
  • 修回日期:  2022-12-22
  • 刊出日期:  2023-03-22

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