1、 鲍江舰 等/fliL 基因显著影响艰难拟梭菌运动功能及产孢能力 Chinese Journal of Biotechnology http:/ Apr.25,2023,39(4):1578-1595 DOI:10.13345/j.cjb.220765 2023 Chin J Biotech,All rights reserved 资助项目：国家自然科学基金(32170134，32160015，U1812403)；贵州省自然科学基金(20201Z067，20191441)；贵州医科大学优秀青年人才计划(2022101)This work was supported by the Nationa
2、l Natural Science Foundation of China(32170134,32160015,U1812403),the Guizhou Provincial Natural Science Foundation(20201Z067,20191441),and the Excellent Young Talents Plan of Guizhou Medical University(2022101).*Corresponding authors.E-mail:CUI Guzhen,;HONG Wei, Received:2022-09-24;Accepted:2022-12
3、-06;Published online:2022-12-09 1578 生物工程学报 fliL 基因显著影响艰难拟梭菌运动功能及产孢能力 鲍江舰1,2，杨君仪2，邵瑞瑞2，张婷2，廖健4，程玉梅5，官志忠2,6，齐晓岚2,3，陈峥宏1，洪伟2,3*，崔古贞1*1 贵州医科大学基础医学院 贵州省普通高等学校病原生物学特色重点实验室，贵州 贵阳 550004 2 贵州医科大学 地方病与少数民族疾病教育部重点实验室 贵州省医学分子生物学重点实验室，贵州 贵阳 550001 3 省部共建地方病及民族区域性疾病防控协同创新中心，贵州 贵阳 550001 4 贵州医科大学口腔医学院 附属口腔医院，贵州
4、贵阳 550001 5 贵州医科大学附属医院综合 ICU，贵州 贵阳 550001 6 贵州医科大学病理科 贵州医科大学附属医院，贵州 贵阳 550001 鲍江舰,杨君仪,邵瑞瑞,张婷,廖健,程玉梅,官志忠,齐晓岚,陈峥宏,洪伟,崔古贞.fliL 基因显著影响艰难拟梭菌运动功能及产孢能力J.生物工程学报,2023,39(4):1578-1595.BAO Jiangjian,YANG Junyi,SHAO Ruirui,ZHANG Ting,LIAO Jian,CHENG Yumei,GUAN Zhizhong,QI Xiaolan,CHEN Zhenghong,HONG Wei,CUI Guz
5、hen.The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficileJ.Chinese Journal of Biotechnology,2023,39(4):1578-1595.摘 要：鞭毛基底体相关 FliL 家族蛋白(flagellar basal body-associated FliL family protein,fliL)基因编码 FliL 蛋白，FliL 是一种与鞭毛基体相结合的单跨膜蛋白。为研究艰难拟梭菌 fliL 基因功能，使用非等
6、长同源臂偶联等位交换(allele-coupled exchange,ACE)方法成功构建了fliL基因缺失(fliL)和回补(:fliL)突变株，研究突变菌株与野生型菌株(CD630)生长曲线、抗生素敏感性、pH 耐受性、运动能力及产孢能力等表型的差异。结果显示，菌株 fliL 生长速率及最大生物量均小于菌株CD630，:fliL 回补菌株生长情况回复至野生型。与 CD630 菌株相比，fliL 对阿莫西林、氨苄青霉素、诺氟沙星的敏感性提高，对卡那霉素、四环素敏感性降低，:fliL 抗生素敏感性部分回复至野生型水平。与 CD630 菌株相比，fliL 游泳运动能力显著降低，:fliL 运动能
7、力超越野生型菌株CD630。相比菌株 CD630，菌株 fliL 在 pH 值为 5 时耐受能力显著提高，在 pH 值为 9 时，耐受能力显著降低。除此之外，fliL 产孢能力较 CD630 显著降低，:fliL 产孢能力部分恢复。以上结果表明，艰难拟梭菌 fliL 基因与其运动能力、抗生素敏感性、环境耐受能力和产孢能力密切相关，可能进一步影响艰难拟梭菌菌株的致病力。医药生物技术 鲍江舰 等/fliL 基因显著影响艰难拟梭菌运动功能及产孢能力 ：010-64807509： 1579 关键词：艰难拟梭菌；非等长同源臂偶联等位交换；鞭毛；fliL；突变株表型 The fliL gene signi
8、ficantly affects the motility and sporulation abilities of Clostridioides difficile BAO Jiangjian1,2,YANG Junyi2,SHAO Ruirui2,ZHANG Ting2,LIAO Jian4,CHENG Yumei5,GUAN Zhizhong2,6,QI Xiaolan2,3,CHEN Zhenghong1,HONG Wei2,3*,CUI Guzhen1*1 Key Laboratory of Microbiology and Parasitology of Education Dep
9、artment of Guizhou,School of Basic Medical Science,Guizhou Medical University,Guiyang 550004,Guizhou,China 2 Key Laboratory of Medical Molecular Biology of Guizhou Province,Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education,Guizhou Medical University,Guiyang 550001,Guizhou,China 3 C
10、ollaborative Innovation Center for Prevention and Control of Endemic and Ethnic Regional Diseases Co-Constructed by the Province and Ministry,Guiyang 550001,Guizhou,China 4 Affiliated Stomatological Hospital,School of Stomatology,Guizhou Medical University,Guiyang 550001,Guizhou,China 5 Department o
11、f Critical Care Medicine,the Affiliated Hospital of Guizhou Medical University,Guiyang 550001,Guizhou,China 6 The Affiliated Hospital of Guizhou Medical University,Department of Pathology,Guizhou Medical University,Guiyang 550001,Guizhou,China Abstract:Flagella are the main motility structure of Clo
12、stridioides difficile that affects the adhesion,colonization,and virulence of C.difficile in the human gastrointestinal tract.The FliL protein is a single transmembrane protein bound to the flagellar matrix.This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-ass
13、ociated FliL family protein(fliL)on the phenotype of C.difficile.The fliL gene deletion mutant(fliL)and its corresponding complementary strains(:fliL)were constructed using allele-coupled exchange(ACE)and the standard molecular clone method.The differences in physiological properties such as growth
14、profile,antibiotic sensitivity,pH resistance,motility,and spore production ability between the mutant and wild-type strains(CD630)were investigated.The fliL mutant and the:fliL complementary strain were successfully constructed.After comparing the phenotypes of strains CD630,fliL,and:fliL,the result
15、s showed that the growth rate and maximum biomass of fliL mutant decreased than that of CD630.The fliL mutant showed increased sensitivity to amoxicillin,ampicillin,and norfloxacin.Its sensitivity to kanamycin and tetracycline antibiotics decreased,and the antibiotic sensitivity partially returned t
16、o the level of CD630 strain in the:fliL strain.Moreover,the motility was significantly reduced in the fliL mutant.Interestingly,the motility of the:fliL strain significantly increased even when compared to that of the CD630 strain.Furthermore,the pH tolerance of the fliL mutant significantly increased or decreased at pH 5 or 9,respectively.Finally,the sporulation ability of fliL mutant reduced considerably compared to the CD630 strain and recovered in the:fliL strain.We conclude that the deletio
