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

鲍曼不动杆菌是一种革兰氏阴性杆菌，由于其对抗生素的高度耐药性而引起人们的极大兴趣。虽然鲍曼不动杆菌对抗生素的耐药性是多种后天因素和内在因素综合作用的结果，但属于耐药结节分裂(RND)家族的质子梯度泵被认为是造成鲍曼不动杆菌内在耐药性的最重要因素。RND泵是一个古老的蛋白质家族，它们的同源物在所有三个王国都有报道。RND泵在革兰氏阴性细菌中被研究得最多，主要是因为它们能够将各种抗菌素泵出两层膜。由于其古老的性质，人们一致认为抗生素的外排不是它们的主要作用，然而RND泵的生理功能(S)仍然未知。在这一应用中，我们建议研究鲍曼不动杆菌的两个RND泵，AdeIJK和AdeFGH，以确定它们的自然功能。我们实验室的初步数据表明，脂肪酸的扰动会导致AdeIJK泵的上调。此外，我们还观察到，AdeIJK Pump的转录抑制因子Aden的缺失导致了近250个不同基因的表达变化。这表明，AdeIJK Pump可能是鲍曼艾美耳氏菌失去亚丁所致的反应的一部分。鉴定和鉴定与AdeIJK共调控的基因将为了解AdeIJK泵的自然功能(S)提供重要线索。同样，我们已经分离出过表达AdeFGH Pump的鲍曼不动杆菌突变体。这些突变体在CSRA中包含一个突变，编码碳储存调节蛋白。CSRA是一种mRNA结合蛋白，在大肠杆菌中抑制几个静止期过程并激活指数期功能。它还调节涉及中枢碳代谢、次生代谢物代谢、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和氮代谢与鲍曼不动杆菌AdeFGH表达之间的联系。我们将采用一种独特的方法，在一种对抗生素敏感的鲍曼不动杆菌环境分离株中进行拟议的研究。我们在鲍曼不动杆菌环境分离株中进行这些研究的方法可能会对AdeIJK和AdeFGH泵的生理功能提供更相关的发现。这项工作将有助于更好地理解鲍曼不动杆菌的代谢途径如何影响RND外排泵的表达。

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.