Salmonella, colonization resistance, and fructose-asparagine

沙门氏菌、定植抗性和果糖天冬酰胺

• 批准号: 8966010
• 负责人: Brian M Ahmer
• 金额: $ 49.66万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966010
• 关键词: Affect; Agriculture; Ammonia; Antibiotic Resistance; Antibiotics; Asparagine; Aspartate; Attenuated; Bacteria; Binding Sites; Biochemical; Bioinformatics; Biological Assay; Carbon; Catabolism; Characteristics; Cleaved cell; Communities; Cytoplasm; DNA Binding; Defect; Dependence; Development; Disease; Drug Targeting; Elderly; Engineering; Enzymatic Biochemistry; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Escherichia coli; Exhibits; Experimental Models; Family; Feedback; Food; Foundations; Fructose; Future; Genetic; Glucose; Growth; Health; Human; Immunocompromised Host; Inbred CBA Mice; Individual; Infection; Inflammation; Intestines; Lactobacillus reuteri; Lead; Learning; Mammals; Measures; Metabolism; Metagenomics; Methods; Microbe; Modeling; Mus; Nutrient; Operon; Organism; Oxidants; Oxidoreductase; Pathway interactions; Phenotype; Phosphotransferases; Probiotics; Reaction; Regulation; Reporting; Resistance; Respiration; Respiratory Burst; Role; SPI1 gene; Salmonella; Salmonella enterica; Salmonella infections; Salmonella typhimurium; Serotyping; Severities; Side; Source; Streptomycin; System; Systems Biology; Testing; Therapeutic; Translating; Vaccines; Virulence; Weight; asparaginase; design; fitness; foodborne pathogen; gene product; gut microbiota; high throughput screening; indexing; interest; member; microbial community; microbiota; microorganism; mutant; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; pathogen; periplasm; prevent; promoter; resistant strain; small molecule; small molecule inhibitor; therapeutic target; transcription factor

 DESCRIPTION (provided by applicant): Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting humans and agriculture. It has long been thought that nutrient utilization systems of Salmonella would not make effective drug targets because there are simply too many nutrients available to Salmonella in the intestine. However, we have discovered that during growth in the inflamed intestine Salmonella relies heavily on a single nutrient - fructose-asparagine (F-Asn), which is present at high concentrations in human foods. Mutants that cannot acquire F-Asn are severely attenuated suggesting that F-Asn is the primary nutrient utilized by Salmonella during inflammation. No other organism has been reported to synthesize or utilize this compound, although we suspect that a few other pathogens and members of the normal gut microbiota can utilize it. The apparent lack of F-Asn utilization pathways in mammals and most other bacteria suggests a specific and potent therapeutic target for Salmonella. The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1- SPI2- or ttrA mutants, respectively). We hypothesize that if Salmonella can initiate inflammation (or enters a gut that is already slightly inflamed), it can begin tetrathionae respiration during F-Asn catabolism and thereby outcompete the normal microbiota, which are doubly compromised by the inflammation and their ability to only ferment (but not respire) F-Asn. We will test this central postulate and build the foundation for two types of therapeutics to block Salmonella acquisition of F-Asn. In our first specific aim, we will investigate the role of a asparaginase (FraE), kinase (FraD) and deglycase (FraB) in F-Asn utilization. Through biochemical characterization of the individual reactions catalyzed by these Fra enzymes and development of high-throughput assays, we expect to facilitate future screens that will identify small molecule inhibitors of these enzymes. We hypothesize that the FraR transcription factor is a repressor. Therefore, preventing its release from the fra operon promoter would also be of therapeutic interest. We propose to determine the natural inducer of FraR and determine the DNA binding sites of FraR in the fra operon. In the second aim, we plan to employ a combination of metagenomics, selective growth in the presence of F-Asn, and bioinformatics to test the idea that in healthy gut communities there are select members of the microbiota that utilize F-Asn and prevent Salmonella from acquiring this nutrient. Finally, we expect our findings on the enzymology and regulation of F-Asn utilization in Salmonella, and possible competing intestinal microbes, to inform our efforts to design new probiotic bacteria that can reduce the severity and duration of Salmonella infection in mice. Overall, our efforts will lead to a better understanding of Salmonella growth in the inflamed intestine and to novel therapeutics.

 描述（由适用提供）：肠道沙门氏菌鼠伤寒（沙门氏菌）是影响人类和农业的最重要的饮食传播病原体之一。长期以来，人们一直认为沙门氏菌的营养利用率不会产生有效的药物靶标，因为肠道中的沙门氏菌根本无法使用。但是，我们发现，在发炎的肠道沙门氏菌的生长过程中，很大程度上取决于单一营养 - 果糖 - 天冬酰胺（F-ASN），该果糖 - 天冬酰胺（F-ASN）以高浓度的人类食品存在。无法获得F-ASN的突变体严重减弱，表明F-ASN是炎症过程中沙门氏菌利用的主要营养素。尚无其他生物体来合成或利用该化合物，尽管我们怀疑其他一些病原体和正常肠道微生物群的成员可以利用它。哺乳动物和大多数其他细菌的F-ASN利用途径的外观缺乏表明沙门氏菌的特定且潜在的治疗靶标。仅当沙门氏菌可以引发炎症并使用四硫酸作为末端电子受体进行厌氧呼吸时，编码F-ASN利用率FRA的基因座才能提供优势（在SPI1-SPI1-SPI22或TTRA突变体中丢失了FRA表型）。我们假设，如果沙门氏菌可以在我们的第一个特定目的中启动，我们将研究天冬氨酸酶（FRAE），激酶（FRAD）和Degl​​ycase（frab）在F-ASN利用中的作用。通过这些FRA酶催化的个体反应的生化表征和高通量测定的开发，我们期望促进未来的筛选，这些筛选将鉴定这些酶的小分子抑制剂。我们假设FRAR转录因子是复制器。因此，防止其从FRA操纵子启动子中释放也是理论上的。我们建议确定Frar的天然诱导剂，并确定Fraoperon中Frar的DNA结合位点。在第二个目的中，我们计划采用宏基因组学，在F-ASN存在下选择性增长以及生物信息学的选择，以测试以下观点：在健康的肠道群落中，有菌群的精选成员使用F-ASN并防止沙门氏菌吸收这种营养素。最后，我们期望我们关于沙门氏菌中F-ASN利用的酶学和调节的发现，以及可能的肠道微生物，以告知我们为设计新的益生菌细菌的努力，以减少小鼠沙门氏菌感染的严重程度和持续时间。总体而言，我们的努力将使对发炎的肠和新型治疗中的沙门氏菌生长更好地了解。