D-Amino acid and tellurite resistance in NIAID Category A bacterial pathogens

NIAID A 类细菌病原体中的 D-氨基酸和亚碲酸盐耐药性

• 批准号: 7495611
• 负责人: Rodney A. Welch
• 金额: $ 21.63万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495611
• 关键词: Accounting; Amino Acids; Antibiotic Resistance; Antibiotics; Attenuated; Bacillus (bacterium); Bacillus anthracis; Bacteria; Bacterial Genes; Carbon; Categories; Class; Commit; Complex; Condition; Containment; Culture Media; Cysteine; D-Amino Acid Dehydrogenase; D-Serine dehydratase; Disinfectants; Engineering; Escherichia coli; Event; Francisella tularensis; Generations; Genes; Genetic; Goals; Growth; Homologous Gene; Individual; Infection; Laboratories; Learning; Local Anti-Infective Agents; Mediating; Metabolism; Methionine; Methodology; Methods; National Institute of Allergy and Infectious Disease; Nitrogen; Numbers; Nutrient; Organism; Pathogenesis; Phenotype; Phenylalanine; Physiology; Plants; Preparation; Process; Reagent; Recombinant Vaccines; Recombinants; Resistance; Role; Screening procedure; Serine; Signal Transduction; Solid; Source; Standards of Weights and Measures; System; Testing; Time; Toxic effect; Tryptophan; Tyrosine; Urinary tract; Uropathogenic E. coli; Vaccines; Work; Yeasts; Yersinia pestis; biodefense; experience; genetic manipulation; insight; interest; mutant; pathogen; pathogenic bacteria; potassium tellurate(IV); research study; resistance mechanism; tellurite; trait

DESCRIPTION (provided by applicant): With a few exceptions, the metabolism and possible toxicity of D-amino acids for bacteria is not well understood. We will investigate an array of potentially directly selectable resistance markers to different D-amino acids for use in genetic manipulation of select agent exempt strains of the three NIAID class A agents, Yersinia pestis, Francisella tularensis and Bacillus anthracis. In order to simultaneously detoxify and use D-serine as a carbon and nitrogen source in the urinary tract, uropathogenic strains of Escherichia coli possess the D-serine deaminase locus, dsdCXA. We have intensively studied this system and propose to introduce recombinant versions of the dsdXA genes or homologs into the different class A biodefense agents in order to assess the utility of D-serine deaminase (DsdA) as a non-antibiotic, directly selectable marker. Our preliminary results indicate that Y. pestis is sensitive to D-serine when grown in complex media. We will also assess if D-tyrosine, D-cysteine, D-methionine, D-phenylalanine and D-tryptophan will be inhibit growth of the different agents in complex media and modified versions of their respective defined media. There are a variety of known dissimilatory mechanisms in E. coli and Bacillus subtilus for these particular inhibitory amino acids. We will investigate the ability of known dissimilatory genes to provide a directly selectable phenotype. As an alternative approach, we will assess if recombinant versions of fungal or mammalian derived D-amino acid oxidase genes can be used for direct selection of resistance to single D-amino acids or cocktails of D-amino acids. Our search for directly selectable markers has also led to an examination of tellurite resistance. There are at least five different bacterial gene systems identified that confer such resistance. Our preliminary results indicate that B. anthracis and Y. pestis are sensitive to potassium tellurite in complex media. We will attempt to engineer tellurite resistance markers into the three species. Our exploration of sensitivity and resistance mechanisms for D-amino acids and tellurite in this R21 project is expected to provide multiple avenues for generation of new selective markers in these three agents. Our work is very likely to provide insights into how to engineer similar traits in additional bacterial pathogens of biodefense interest.

描述（由申请人提供）： 除少数例外，D-氨基酸对细菌的代谢和可能的毒性尚不清楚。我们将研究一系列针对不同 D-氨基酸的潜在直接选择性抗性标记，用于对三种 NIAID A 类病原体（鼠疫耶尔森氏菌、土拉弗朗西斯氏菌和炭疽杆菌）的选择试剂豁免菌株进行基因操作。为了同时解毒并利用 D-丝氨酸作为泌尿道中的碳源和氮源，尿路致病性大肠杆菌菌株拥有 D-丝氨酸脱氨酶基因座 dsdCXA。我们深入研究了该系统，并建议将 dsdXA 基因或同源物的重组版本引入不同的 A 类生物防御剂中，以评估 D-丝氨酸脱氨酶 (DsdA) 作为非抗生素、直接选择标记的效用。我们的初步结果表明，鼠疫耶尔森氏菌在复杂培养基中生长时对 D-丝氨酸敏感。 我们还将评估 D-酪氨酸、D-半胱氨酸、D-蛋氨酸、D-苯丙氨酸和 D-色氨酸是否会抑制不同试剂在复杂培养基及其各自限定培养基的改良版本中的生长。对于这些特定的抑制性氨基酸，大肠杆菌和枯草芽孢杆菌中有多种已知的异化机制。我们将研究已知的异化基因提供直接选择表型的能力。作为一种替代方法，我们将评估真菌或哺乳动物来源的 D-氨基酸氧化酶基因的重组版本是否可用于直接选择对单一 D-氨基酸或 D-氨基酸混合物的抗性。 我们对直接选择标记的搜索也导致了对亚碲酸盐抗性的检查。至少有五种不同的细菌基因系统被鉴定出具有这种抗性。我们的初步结果表明，炭疽杆菌和鼠疫杆菌对复杂培养基中的亚碲酸钾敏感。我们将尝试将亚碲酸盐抗性标记设计到这三个物种中。 我们在此探索 D-氨基酸和亚碲酸盐的敏感性和耐药机制 R21 项目预计将为在这三种药物中生成新的选择性标记提供多种途径。我们的工作很可能为如何在具有生物防御意义的其他细菌病原体中设计类似特征提供见解。