Function and inhibition of multidrug efflux systems in Campylobacter

 描述(申请人提供)：空肠弯曲杆菌是全球腹泻的主要细菌原因，对临床上重要的抗生素越来越耐药。在已知的弯曲杆菌耐药机制中，多药外排系统CmeABC(一种RND类型的外排转运体)是一个重要的角色，它对结构多样的抗生素和有毒化合物具有耐药性。此外，CmeABC在胆汁抵抗中起着关键作用，对弯曲杆菌在肠道的定植是必不可少的。最近发现氧化应激的反应调节因子COSR作为cmeABC的抑制因子，调节cmeABC在氧化应激中的表达。这些结果有力地表明，氧化应激是一种先前未知的调节CmeABC功能的生理信号，COSR介导的对氧化应激的反应可能对空肠弯曲菌的适应至关重要。空肠弯曲菌是一种在宿主的传播和感染过程中经历氧化应激的微生物。尽管有强有力的初步证据，但氧化应激与COSR-CmeABC途径之间相互作用的详细机制以及这种相互作用在抗生素和氧化应激抵抗中的作用仍不清楚。为了控制耐药弯曲杆菌，我们最近开发了一种新的策略，利用抗cmeABC肽核酸(PNA)来抑制cmeABC的表达。这一方法被发现能有效地增强弯曲杆菌对抗生素的敏感性，这表明针对cmeABC的反义PNA是对抗耐药弯曲杆菌的一种有前途的方法。这项应用是基于这些令人兴奋的发现，并将追求两个特定的目标：i)确定氧化应激与COSR-CmeABC途径相互作用的机制，并确定这种相互作用在弯曲杆菌对抗生素和氧化应激适应中的作用；ii)在动物模型中确定抗cmeABC PNA在防止抗生素耐药突变株的出现和增强抗生素对弯曲菌的作用方面的有效性。该提案在概念和技术上都是创新的，因为它解决了氧化应激反应和抗生素外排系统之间的一个新兴主题，并通过靶向CmeABC开发了一种新的抗弯曲菌方法。这项拟议的工作将极大地推进氧化应激感知和抵抗是细菌病原体中抗生素外排系统的一种常见生理功能的概念，并将开发一种有效的手段来扩大现有抗生素对耐药弯曲杆菌的应用。此外，该项目建立的技术平台可能适用于其他耐药病原体的控制。

 DESCRIPTION (provided by applicant): Campylobacter jejuni is a leading bacterial cause of diarrhea worldwide and is increasingly resistant to clinically important antibiotics. Among the known mechanisms involved in antibiotic resistance in Campylobacter, the multidrug efflux system CmeABC (an RND-type efflux transporter) is a significant player and confers resistance to structurally diverse antibiotics and toxic compounds. Additionally, CmeABC plays a critical role in bile resistance and is essential for Campylobacter colonization in the intestinal tract. Mot recently it was found that CosR, a response regulator for oxidative stress, serves as a repressor for cmeABC and modulates the expression of cmeABC in response to oxidative stress. These results strongly suggest that oxidative stress is a previously unidentified physiological signal modulating the function of CmeABC and the CosR-mediated response to oxidative stress may be important for the adaptation of C. jejuni, a microaerobic organism that experiences oxidative stress during transmission and infection of a host. Despite strong preliminary evidence, the detailed mechanisms of the interplay between oxidative stress and the CosR- CmeABC pathway and the role of this interaction in antibiotic and oxidative stress resistance are still unknown. To control antibiotic-resistant Campylobacter, we recently developed a novel strategy that utilizes anti-cmeABC peptide nucleic acid (PNA) to inhibit the expression of cmeABC. This approach was found to be effective in sensitizing Campylobacter to antibiotics, suggesting that antisense PNA targeting cmeABC is a promising approach for combating antibiotic-resistant Campylobacter. This application is based on these exciting findings and will pursue two Specific Aims i) to determine the mechanisms by which oxidative stress interacts with the CosR-CmeABC pathway and define the role of this interaction in Campylobacter adaptation to antibiotic and oxidative stresses, and ii) to determine the efficiency of anti-cmeABC PNA in preventing the emergence of antibiotic resistant mutants and potentiating antibiotics against Campylobacter in animal models. The proposal is innovative both conceptually and technically as it addresses an emerging theme at the interface between oxidative stress response and antibiotic efflux systems and develops a novel anti-Campylobacter approach by targeting CmeABC. The proposed work will significantly advance the concept that oxidative stress sensing and resistance is a common physiological function of antibiotic efflux systems in bacterial pathogens and will develop an effective mean to extend the utility of existing antibiotic against drug-resistant Campylobacter. Furthermore, the technical platform established in this project can be potentially adapted for the control of other antibiotic-resistant pathogens.