Millanao Ana R, Mora Aracely Y, Saavedra Claudia P, Villagra Nicolás A, Mora Guido C, Hidalgo Alejandro A
Escuela de Química y Farmacia, Facultad de Medicina, Universidad Andres Bello, Santiago, Chile.
Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Front Microbiol. 2020 Mar 20;11:428. doi: 10.3389/fmicb.2020.00428. eCollection 2020.
Ciprofloxacin is the choice treatment for infections caused by Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn transposon mutants of . Typhi were screened. Typhi ::EZ-Tn mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene . Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the mutant. Expression of increased four times in the null mutant compared to WT strain. To understand the relationship between the expression of and , a strain with the gene under control of the tetracycline-inducible P promoter was created, to modulate expression. Induction of decreased expression of , at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the mutant, compared to WT strain. In addition, expression of and genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the mutant. Further studies indicate that deletion of decreases susceptibility to CIP, while deletion of gene increases susceptibility CIP. Our findings indicate that inactivation promotes expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.
环丙沙星是治疗伤寒杆菌感染的首选药物，然而，已有报道称该病原体对环丙沙星的敏感性降低。考虑到新抗菌药物的批准减少和耐药性危机，应对这一问题的一种策略是寻找新的靶点，以增强已批准抗菌药物的抗菌活性。为了寻找对环丙沙星敏感性增加的突变体，筛选了3216个伤寒杆菌的EZ-Tn转座子突变体。通过琼脂扩散法和MIC测定法确认了对环丙沙星敏感的伤寒杆菌::EZ-Tn突变体。对携带EZ-Tn转座子插入的基因进行了测序。使用定点诱变产生了中断基因的缺失突变体以及诱导型遗传构建体，以证实表型。分别使用SDS-PAGE和实时PCR评估蛋白质和基因的表达。在筛选中发现了5个对环丙沙星敏感性增加的突变体。第一个得到确认的突变体是谷氨酰胺合成酶编码基因。外膜蛋白分析显示，在该突变体中，环丙沙星和萘啶酸流入通道OmpF增加。与野生型菌株相比，在该缺失突变体中，该基因的表达增加了四倍。为了了解该基因与OmpF表达之间的关系，构建了一个在四环素诱导型P启动子控制下带有该基因的菌株，以调节该基因的表达。该基因的诱导降低了OmpF的表达，同时降低了对环丙沙星的敏感性。与野生型菌株相比，在该突变体中，sRNA MicF（OmpF的负调节因子）的表达降低至四分之一。此外，在该突变体中，该基因和该基因（编码可能正向调节OmpF的双组分系统NtrC/B）的表达增加。进一步的研究表明，该基因的缺失降低了对环丙沙星的敏感性而该基因的缺失增加了对环丙沙星的敏感性。我们的研究结果表明，该基因失活促进了该基因的表达，这转化为OmpF蛋白增加，促进了环丙沙星的进入，从而通过两种可能的机制增加了对环丙沙星的敏感性。
