Determining physiological role(s) of Resistance-Nodulation-Division efflux pumps of Acinetobacter baumannii

确定鲍曼不动杆菌抵抗结瘤分裂外排泵的生理作用

• 批准号: RGPIN-2021-02902
• 负责人: Kumar, Ayush
• 金额: $ 4.23万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742274
• 关键词: Determining; physiological; role; Resistance; Nodulation

Acinetobacter baumannii is a Gram-negative pathogen and an organism of significant interest due to its high resistance to antibiotics. Although A. baumannii's resistance to antibiotics is a result of a combination of various acquired and intrinsic factors, proton-gradient-dependent pumps belonging to the Resistance-Nodulation-division (RND) family are considered the most important factors contributing to the intrinsic antibiotic resistance of A. baumannii. RND pumps are an ancient family of proteins and their homologs have been reported across all three kingdoms. RND pumps have been most studied in Gram-negative bacteria primarily due to their ability to pump out a variety of antimicrobials across the two membranes. Due to their ancient nature, there is a consensus that the efflux of antibiotics is not their primary role, however physiological function(s) of RND pumps remain unknown. In this application, we propose to study two RND pumps of A. baumannii, AdeIJK and AdeFGH to determine their natural functions. Preliminary data from our laboratory suggest that fatty acid perturbations can result in the upregulation of AdeIJK pump. Further, we have also observed that the deletion of AdeN, the transcriptional repressor of AdeIJK pump, results in an altered expression of almost 250 different genes. This suggests that perhaps AdeIJK pump is part of a response in A. baumannii that results from the loss of AdeN. Identifying and characterizing genes that are co-regulated with AdeIJK will provide important clues about the natural function(s) of AdeIJK pump. Similarly, we have isolated mutants of A. baumannii that overexpress AdeFGH pump. These mutants contain a mutation in csrA that encodes for the Carbon Storage Regulator protein. CsrA, an mRNA binding protein, represses several stationary phase processes and activates exponential-phase functions in E. coli. It also modulates the regulation of genes involved in central carbon metabolism, secondary metabolite metabolism, motility, biofilm formation, as well as virulence. We are therefore proposing to study the link between central carbon as well nitrogen metabolism and the expression of AdeFGH in A. baumannii. We will employ a unique approach and carry out the proposed studies in an antibiotic susceptible environmental isolate of A. baumannii. Our approach of carrying out these studies in an environmental isolate of A. baumannii is likely to provide more relevant findings on the physiological functions of AdeIJK and AdeFGH pumps. This work will lead to a better understanding of how the expression of RND efflux pumps may be influenced by metabolic pathways in A. baumannii.

鲍曼不动杆菌是一种革兰氏阴性病原体，由于其对抗生素的高抗性，是一种重要的生物体。虽然A.鲍曼不动杆菌对抗生素的耐药性是多种获得性因素和内在因素共同作用的结果，属于耐药-结瘤-分裂（Resistance-Nodulation-Division，RND）家族的质子梯度依赖性泵被认为是导致鲍曼不动杆菌内在抗生素耐药性的最重要因素。鲍曼不动杆菌。RND泵是一个古老的蛋白质家族，它们的同系物在所有三个王国中都有报道。RND泵在革兰氏阴性菌中研究最多，主要是因为它们能够跨两层膜泵出各种抗菌剂。由于其古老的性质，有一个共识，即抗生素的外排不是其主要作用，然而RND泵的生理功能仍然未知。在这个应用中，我们提出了研究两个RND泵的A。baumannii、AdeIJK和AdeFGH来确定它们的天然功能。来自我们实验室的初步数据表明，脂肪酸扰动可导致AdeIJK泵的上调。此外，我们还观察到，亚丁（AdeIJK泵的转录抑制因子）的缺失导致近250种不同基因的表达改变。这表明AdeIJK泵可能是A.鲍曼不动杆菌，其由亚丁的缺失引起。识别和表征与AdeIJK共调节的基因将提供关于AdeIJK泵的天然功能的重要线索。同样，我们分离出了A.过表达AdeFGH泵的鲍曼不动杆菌。这些突变体在编码碳储存调节蛋白的csrA中含有突变。CsrA是一种mRNA结合蛋白，在E.杆菌它还调节参与中心碳代谢、次级代谢物代谢、运动性、生物膜形成以及毒力的基因的调节。因此，我们建议研究中心碳以及氮代谢和AdeFGH在A.鲍曼不动杆菌。我们将采用一种独特的方法，在一种抗生素敏感的环境分离株中进行拟议的研究。鲍曼不动杆菌。我们的方法进行这些研究在一个环境分离的A。鲍曼不动杆菌可能提供关于AdeIJK和AdeFGH泵的生理功能的更多相关发现。这项工作将导致更好地了解RND外排泵的表达可能受到代谢途径的影响。鲍曼不动杆菌。