Determination of Porphyromonas antimicrobial resistance mechanisms by Tn-Seq

牙龈卟啉单胞菌是一种厌氧、革兰氏阴性细菌，是牙周炎发病机制中的一种重要微生物。抗生素被用来治疗严重的牙周炎，最近有报道称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类化合物，对许多厌氧细菌都有抗菌活性，包括牙龈假单胞菌。据报道，对甲硝唑的耐药率高达22%，但对耐药机制知之甚少。在这项提案中，我将使用转座子突变来鉴定与甲硝唑耐药性产生有关的基因和基因网络。在我的前期工作中，我使用基于Mariner的转座子系统在不同菌株的牙龈假单胞菌中创建了两个转座子突变体文库。这种转座子系统与之前牙龈假单胞菌中的转座子文库相比具有优势，这是因为Mariner能够随机插入，从而创建在整个基因组中插入的高度饱和的文库。利用这个文库，我已经鉴定了以前未被识别的牙龈假单胞菌的色素突变。有趣的是，这些色素沉着缺陷的PG突变体中的一些对甲硝唑具有耐药性。在这项建议中，我将首先通过对我分离的色素突变株进行补充和鉴定来建立对甲硝唑耐药的色素沉着的RLE。我使用的转座子适用于大规模平行测序，通过一种名为TN-SEQ的技术来鉴定文库中突变的相对适合度。为了发现更多与甲硝唑耐药性有关的非色素相关基因，我将使用TN-SEQ对整个文库进行筛选，以确定在接触不同浓度的甲硝唑时，哪些突变会被浓缩或消除。最后，我将对牙周病患者的牙龈假单胞菌进行临床采样，并对AIMS 1和2中确定的甲硝唑耐药基因进行定向基因测序，以确定在临床耐药牙周炎患者中是否发现与体外对甲硝唑耐药相关的突变。该项目有望为揭示牙龈假单胞菌对一种重要抗菌剂的耐药性机制提供新的线索。在这个过程中，我将开发新的工具和学习技术，这些工具和学习技术有可能应用于牙龈假单胞菌毒力的多个方面，这将为我未来在口腔微生物学研究中做好准备。

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.