Determination of Porphyromonas antimicrobial resistance mechanisms by Tn-Seq

描述（申请人提供）：牙龈卟啉单胞菌是一种革兰氏阴性厌氧菌，是牙周炎发病机制中的重要微生物。抗生素用于治疗严重的牙周炎，最近临床分离的牙龈假单胞菌对抗生素的耐药性越来越强。甲硝唑是一种硝基咪唑，对许多厌氧菌，包括牙龈假单胞菌有活性。据报道，目前对甲硝唑的耐药性高达22%，但对耐药机制知之甚少。在本提案中，我将使用转座子诱变来鉴定参与甲硝唑抗性发展的基因和基因网络。在我的初步工作中，我使用基于mariner的转座子系统在不同的牙龈假单胞菌菌株中创建了两个转座子突变文库。该转座子系统与先前的牙龈p.p ingivalis转座子文库相比具有优势，因为Mariner能够随机插入，从而在整个基因组中创建高度饱和的插入文库。利用这个文库，我已经发现了以前未被识别的牙龈卟啉卟啉菌色素沉着突变体。有趣的是，其中一些色素缺陷的Pg突变体对甲硝唑有抗性。在本提案中，我将首先通过对我分离的色素沉着突变体进行互补和表征来确定色素沉着在甲硝唑抗性中的作用。我使用的转座子适用于大规模平行测序，用一种叫做n-seq的技术来鉴定文库中突变体的相对适应度。为了发现参与甲硝唑抗性的其他非色素相关基因，我将使用n-seq对整个文库进行筛选，以确定暴露于不同浓度甲硝唑时富集或消除的突变体。最后，我将对牙周病患者的牙龈卟啉单胞菌进行临床取样，并对目的1和目的2中鉴定的甲硝唑耐药基因进行靶向基因测序，以确定临床耐药的牙龈卟啉单胞菌患者中是否存在与甲硝唑体外耐药相关的突变。该项目有望为研究牙龈假单胞菌对一种重要抗菌药物的耐药机制提供新的线索。在这个过程中，我将开发新的工具和学习技术，这些技术有可能应用于牙龈假单胞菌毒力的多个方面，这将为我未来的口腔微生物研究做好准备。

DESCRIPTION (provided by applicant): Porphyromonas gingivalis, an anaerobic, Gram-negative bacterium, is an important organism in the pathogenesis of periodontitis. Antibiotics are used in the treatment of severe periodontitis and recently growing resistance to antibiotics in clinical P.gingivalis isolates has been reported. Metronidazole is a nitroimidazole with activity against many anaerobic bacteria, including P.gingivalis. Resistance to metronidazole has now been reported as high as 22%, but little is known about the mechanisms of resistance. In this proposal, I will use transposon mutagenesis to identify genes and gene networks involved in the development of metronidazole resistance. In my preliminary work, I have created two transposon mutant libraries in different strains of P.gingivalis using a Mariner-based transposon system. This transposon system has advantages compared to prior transposon libraries in P.gingivalis due to ability of Mariner to insert randomly as to create highly saturated libraries with insertions throughout the genome. Using this library, I have already identified previously unrecognized pigmentation mutants of P.gingivalis. Interestingly, some of these pigmentation-defective Pg mutants are resistant to metronidazole. In this proposal, I will first establish the rle of pigmentation in resistance to metronidazole by performing complementation and characterization of the pigmentation mutants I have isolated. The transposon I used is adapted for use with massively-parallel sequencing to identify the relative fitness of mutants in the libray with a technique called Tn-seq. To uncover additional non-pigment related genes involved in metronidazole resistance, I will perform a screen of the entire library using Tn-seq to identify mutants that are either enriched or eliminated on exposure to different concentrations of metronidazole. Finally, I will perform clinical sampling for P.gingivalis from patients with periodontal disease and employ targeted gene sequencing of metronidazole resistance genes identified in Aims 1 and 2, to determine whether mutations associated with in vitro resistance to metronidazole are found in patients with clinically resistant isolates of P.gingivalis. This projec is expected to shed new light on mechanisms of P.gingivalis resistance to an important antimicrobial agent. In the process I will be developing new tools and learning techniques that have the potential to be applied to multiple aspects of P.gingivalis virulence and that will prepar me for a future in oral microbiological research.