Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, USA.
Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, Omaha, NE, USA.
J Antimicrob Chemother. 2020 May 1;75(5):1151-1158. doi: 10.1093/jac/dkz566.
Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic.
To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production.
RNA expression was evaluated using real-time RT-PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting.
Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21-30 min for ST131 isolates compared with <2-23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with -4- to 70-fold for non-ST131 isolates).
Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment.
毒力基因和耐药机制的表达无疑在大流行克隆大肠杆菌 ST131 的成功传播中发挥了作用。孔蛋白下调是一种与抗生素耐药性相关的染色体机制。孔蛋白翻译可以受到 mRNA 半衰期和小 RNA(sRNA)、孔蛋白转录本和 sRNA 伴侣 Hfq 之间相互作用的影响。孔蛋白蛋白翻译的可译性的改变可能会影响大肠杆菌 ST131 分离株在存在抗生素时的适应性和因此的成功。
通过比较大肠杆菌不同 ST 型的 OmpC 和 OmpF 孔蛋白的稳态 RNA 水平、mRNA 半衰期、调节 sRNA 表达和蛋白产量,来确定它们的翻译可变性的差异。
使用实时 RT-PCR 评估 RNA 表达,并用 northern blot 评估 OmpC mRNA 半衰期。通过免疫印迹评估 OmpC、OmpF 和 Hfq 蛋白水平。
ST131 与非 ST131 分离株之间的差异包括:(i)ST131 分离株的 OmpC RNA 和蛋白水平较高,但 OmpC 蛋白水平低于非 ST131 分离株;(ii)OmpC mRNA 半衰期(ST131 分离株为 21-30 分钟,而非 ST131 分离株为 <2-23 分钟);和(iii)sRNA MicC 水平(ST131 分离株为 2-120 倍,而非 ST131 分离株为 -4-70 倍)。
不同大肠杆菌 ST 型的孔蛋白蛋白翻译的可变性涉及不同的机制。这些差异可能为 ST131 型大肠杆菌在面对富含抗生素的环境时提供了选择性优势。