南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (4): 757-764.doi: 10.12122/j.issn.1673-4254.2024.04.18

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肠道硝酸盐对肺炎克雷伯菌生长的影响及其调控机制

谢继臣，马仁惠，李默然，李 蓓，熊莉娜   

  1. 湖北医药学院基础医学院，湖北 十堰 442000；华中科技大学同济医学院附属梨园医院消化内科，湖北 武汉 430077
  • 发布日期:2024-04-29

Effect of intestinal nitrate on growth of Klebsiella pneumoniae and its regulatory mechanism

XIE Jichen, MA Renhui, LI Moran, LI Bei, XIONG Lina   

  1. School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China; Department of Gastroenterology, Liyuan Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430077, China
  • Published:2024-04-29

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摘要： 目的 探讨肠道硝酸盐环境对肺炎克雷伯菌生长的影响及具体调控机制。方法 构建肺炎克雷伯菌硝酸还原酶narG、narZ单基因和双基因敲除株及硝酸盐二元调控系统NarXL基因敲除株；全自动细菌生长分析仪检测菌株在体外有氧硝酸盐环境下的生长曲线；分光光度计法检测细菌厌氧硝酸盐环境下生长曲线；qRT-PCR检测二元调控系统NarXL在厌氧硝酸盐环境中对硝酸还原酶narG、narZ基因表达影响；EMSA和MST分析NarXL在感应利用硝酸盐环境中的具体调控机制；qRT-PCR检测厌氧硝酸盐环境对narG、narZ基因表达的影响；利用竞争实验检测narG、narZ基因在厌氧硝酸盐环境中的生长优势。结果 相比于有氧环境，在厌氧条件下，硝酸盐能够明显促进肺炎克雷伯菌野生株的生长且促进生长的幅度高于narXL基因敲除株；qRT-PCR显示厌氧条件下narXL敲除株中硝酸还原酶相关基因narG、narZ表达显著降低（P<0.0001）；EMSA和MST实验显示NarXL调控子能够直接与narG、narZ启动子区结合；qRT-PCR显示在厌氧硝酸盐环境中硝酸还原酶narG、narZ基因表达量明显升高（P<0.0001，P<0.01）；生长曲线和竞争实验表明硝酸还原酶narG基因敲除株在厌氧硝酸盐环境中生长和竞争生长能力均明显变弱（P<0.01）。结论 肺炎克雷伯菌二元调控系统NarXL可感应肠道硝酸盐浓度变化直接正调控硝酸还原酶基因narG、narZ表达从而促进细菌生长。

关键词: 肺炎克雷伯菌；NarXL；硝酸还原酶；厌氧生长；肠道环境

Abstract: Objective To explore the effect of intestinal nitrates on the growth of Klebsiella pneumoniae and its regulatory mechanisms. Methods K. pneumoniae strains with nitrate reductase narG and narZ single or double gene knockout or with NarXL gene knockout were constructed and observed for both aerobic and anaerobic growth in the presence of KNO3 using an automated bacterial growth analyzer and a spectrophotometer, respectively. The mRNA expressions of narG and narZ in K.pneumoniae in anaerobic cultures in the presence of KNO3 and the effect of the binary regulatory system NarXL on their expresisons were detected using qRT-PCR. Electrophoretic mobility shift assays (EMSA) and MST analysis were performed to explore the specific regulatory mechanisms of NarXL in sensing and utilizing nitrates. Competitive experiments were conducted to examine anaerobic growth advantages of narG and narZ gene knockout strains of K. pneumoniae in the presence of KNO3. Results The presence of KNO3 in anaerobic conditions, but not in aerobic conditions, promoted bacterial growth more effectively in the wild-type K. pneumoniae strain than in the narXL gene knockout strain. In anaerobic conditions, the narXL gene knockout strain showed significantly lowered mRNA expressions of narG and narZ (P<0.0001). EMSA and MST experiments demonstrated that the NarXL regulator could directly bind to narG and narZ promoter regions. The wild-type K.pneumoniae strain in anaerobic cultures showed significantly increased expressions of narG and narZ mRNAs in the presence of KNO3 (P<0.01), and narG gene knockout resulted in significantly attenuated anaerobic growth and competitive growth abilities of K. pneumoniae in the presence of KNO3 (P<0.01). Conclusion The binary regulatory system NarXL of K. pneumoniae can sense changes in intestinal nitrate concentration and directly regulate the expression of nitrate reductase genes narG and narZ to promote bacterial growth.

Key words: Klebsiella pneumoniae; NarXL; nitrate reductase; anaerobic growth; intestinal environment