fliL基因显著影响艰难拟梭菌运动功能及产孢能力

The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile

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(ΔfliL)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ΔfliL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，ΔfliL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，ΔfliL游泳运动能力显著降低，::fliL运动能力超越野生型菌株CD630。相比菌株CD630，菌株ΔfliL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，ΔfliL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。

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

Abstract:

Flagella are the main motility structure of Clostridioides 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-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) 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 profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the::fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and::fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the::fliL strain. Moreover, the motility was significantly reduced in the ΔfliL 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 ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the::fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Key words:Clostridioides difficile;allele-coupled exchange (ACE);flagellum;fliL;phenotypes of mutants