Genetic determinants of antimicrobial peptide resistance in Gram negative bacteri

说明(申请人提供)：抗菌肽是一类很有前途的新型抗菌剂，特别是由于它们对耐常规抗生素的细菌具有强大的活性。到目前为止，多粘菌素是唯一一种对铜绿假单胞菌和鲍曼不动杆菌等多重耐药的革兰氏阴性菌敏感的抗生素。在结构上与环状脂肽不同的抗菌肽多粘菌素目前正处于临床试验的不同阶段，作为全身和局部用药，例如阳离子1-螺旋聚己聚糖和2-片状plectasin。然而，最近的研究表明，抗菌肽在临床上的使用最终会导致细菌对它们产生高水平的耐药性。在最糟糕的情况下，通常由人类内源性抗菌肽控制的细菌可能会导致无法控制的感染。我们在这个项目中的目标是识别和表征细菌对人抗菌肽如阳离子2-页中性粒细胞多肽-1(HNP-1)和阴离子真皮杀菌素-1的内在耐药性的遗传决定因素，作为评估细菌对它们的耐药性的潜在风险和克服这种耐药性的先决条件。在革兰氏阴性菌中，对这类抗菌肽的耐药机制几乎是未知的。为了加速发现抗性决定因素，我们开发了一种基于微阵列的新方法，即监测基因敲除(MGK)。MGK可以同时分析在一种培养中生长的数千个突变株的相对丰度。我们的初步研究表明，这种方法非常有效，使用MGK的初步实验已经确定了以前未知的与HNP-1抗性有关的基因。基于这一强大的方法，我们提出了以下具体目标：(1)鉴定阳离子2-Sheet HNP-1抗性的遗传决定因素；(2)鉴定阴离子皮肤杀菌素抗性的遗传决定因素；以及(3)鉴定与HNP-1和皮肤杀菌素抗性相关的基因。该项目的成功完成将为按计划在后续RO1项目中深入了解细菌对抗菌肽的耐药性机制奠定基础。此外，随着抗菌肽耐药性缺陷的细菌病原体在动物感染中减弱，全面的耐药性决定因素清单可能有助于了解它们在细菌发病机制中的贡献，并可能为新的抗感染策略提供见解。 抗菌肽是一类很有前途的新型抗菌药物，因为它们对耐常规抗生素的细菌具有强大的杀菌活性，目前正处于全身和局部使用的临床试验的不同阶段。因此，了解细菌对这些多肽的天然耐药性是评估临床环境中可能出现的高度耐药细菌并可能制定克服这种耐药性的措施的先决条件。

DESCRIPTION (provided by applicant): Antimicrobial peptides are a promising class of new antibacterial agents, particularly due to their potent activity against bacteria resistant to conventional antibiotics. Only a few such peptides, e.g., polymyxins, have been in clinical practice, and to date polymyxins are the only antibiotic to which multidrug-resistant Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii remain susceptible. Antimicrobial peptides that structurally differ from the cyclic lipopeptide polymyxins are now at various stages of clinical trials as systemic and topical agents, e.g., cationic 1-helical pexiganan and 2-sheet plectasin. However, recent studies have demonstrated that the use of antimicrobial peptides in clinics will eventually induce a high level of bacterial resistance to them. In its worst scenario, bacteria normally controlled by human endogenous antimicrobial peptides may cause unmanageable infections. Our goal in this project is to identify and characterize genetic determinants of bacterial intrinsic resistance to human antimicrobial peptides such as cationic 2-sheet neutrophil peptide-1 (HNP-1) and anionic dermcidin-1, as a prerequisite to assessing the potential risk of bacterial resistance to them and overcoming such resistance. In Gram-negative pathogens, mechanisms of resistance to these classes of antimicrobial peptides are virtually unknown. To expedite the discovery of resistance determinants, we have developed a new microarray-based method, monitoring of gene knockouts (MGK). MGK allows simultaneous analysis of the relative abundance of thousands of mutants grown in a culture. Our preliminary studies demonstrate that this method works very well and a pilot experiment using MGK has identified genes previously unknown to be involved in HNP-1 resistance. Based on this powerful method, we propose the following specific aims: (1) to identify genetic determinants of cationic 2-sheet HNP-1 resistance; (2) to identify genetic determinants of anionic dermcidin resistance; and (3) to characterize genes involved in HNP- 1 and dermcidin resistance. Successful accomplishing of this project will set the stage for in-depth understanding of mechanisms of bacterial resistance to antimicrobial peptides as planned in a follow-up RO1 project. Moreover, as bacterial pathogens defective in antimicrobial peptide resistance are attenuated in animal infections, a comprehensive list of resistance determinants may help understand their contribution to bacterial pathogenesis and may provide insights into new anti-infective strategies. Antimicrobial peptides are a promising class of new antibacterial therapies due to their potent bactericidal activity against bacteria resistant to conventional antibiotics, and they are now at various stages of clinical trials for systemic and topical use. Thus, the understanding of bacterial natural resistance to these peptides is important as a prerequisite to assessing potential emergence of bacteria highly resistant to them in clinical settings and possibly developing measures to overcome such resistance.