Genetic determinants of antimicrobial peptide resistance in Gram negative bacteri

描述（由申请人提供）：抗菌肽是一类有前途的新型抗菌剂，特别是由于其对常规抗生素耐药细菌的有效活性。只有少数这样的肽，例如，多粘菌素已经用于临床实践，并且迄今为止，多粘菌素是唯一的对多重耐药革兰氏阴性细菌如铜绿假单胞菌和鲍氏不动杆菌仍然敏感的抗生素。结构上不同于环脂肽多粘菌素的抗微生物肽现在处于作为全身和局部药剂的临床试验的各个阶段，例如，阳离子1-螺旋Pexiganan和2-折叠Plectasin。然而，最近的研究表明，抗菌肽在临床上的使用最终会诱导细菌对它们产生高水平的耐药性。在最坏的情况下，通常由人类内源性抗微生物肽控制的细菌可能导致无法控制的感染。我们在这个项目中的目标是确定和表征细菌对人类抗菌肽如阳离子2-折叠中性粒细胞肽-1（HNP-1）和阴离子杀皮素-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。在革兰氏阴性病原体中，对这些类别的抗微生物肽的抗性机制实际上是未知的。为了加快抗性决定子的发现，我们开发了一种新的基于微阵列的方法，监测基因敲除（MGK）。MGK允许同时分析培养物中生长的数千种突变体的相对丰度。我们的初步研究表明，这种方法非常有效，使用MGK的试点实验已经鉴定出以前未知的参与HNP-1抗性的基因。基于这种强大的方法，我们提出了以下具体目标：（1）确定阳离子2-折叠HNP-1抗性的遗传决定因素;（2）确定阴离子杀皮素抗性的遗传决定因素;（3）表征参与HNP- 1和杀皮素抗性的基因。该项目的成功完成将为后续RO 1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，由于抗菌肽抗性缺陷的细菌病原体在动物感染中被减弱，因此抗性决定因素的全面列表可能有助于了解它们对细菌发病机制的贡献，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的抗菌治疗，由于其强大的杀菌活性对细菌耐药的常规抗生素，他们现在在临床试验的各个阶段，全身和局部使用。因此，了解细菌对这些肽的天然耐药性是重要的，这是评估在临床环境中可能出现对它们具有高度耐药性的细菌以及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.