Salmonella, colonization resistance, and fructose-asparagine

 性状（由申请方提供）：鼠伤寒沙门氏菌（沙门氏菌）是影响人类和农业的最重要的食源性病原体之一。长期以来，人们一直认为沙门氏菌的营养利用系统不会成为有效的药物靶点，因为肠道中沙门氏菌可利用的营养物质太多了。然而，我们已经发现，在发炎的肠道中生长期间，沙门氏菌严重依赖于单一营养素-果糖-天冬酰胺（F-Asn），其在人类食物中以高浓度存在。不能获得F-Asn的突变体被严重削弱，表明F-Asn是沙门氏菌在炎症期间利用的主要营养素。没有其他生物体已被报道合成或利用这种化合物，虽然我们怀疑，一些其他病原体和成员的正常肠道微生物群可以利用it. The明显缺乏F-Asn利用途径在哺乳动物和大多数其他细菌表明一个特定的和有效的治疗目标沙门氏菌。编码F-Asn利用的基因座fra仅在沙门氏菌可引发炎症并使用连四硫酸盐作为厌氧呼吸的末端电子受体时才具有优势（fra表型分别在沙门氏菌SPI 1-SPI 2-或ttrA突变体中丢失）。我们假设，如果沙门氏菌可以引发炎症（或进入已经轻微发炎的肠道），它可以在F-Asn催化过程中开始四硫堇呼吸，从而胜过正常的微生物群，后者受到炎症和它们仅发酵（但不呼吸）F-Asn的能力的双重损害。我们将测试这一中心假设，并为两种类型的治疗方法奠定基础，以阻止沙门氏菌获得F-Asn。在我们的第一个具体目标中，我们将研究天冬酰胺酶（FraE），激酶（FraD）和脱糖酶（FraB）在F-Asn利用中的作用。通过这些Fra酶催化的各个反应的生物化学表征和高通量检测的发展，我们希望促进未来的筛选，将确定这些酶的小分子抑制剂。我们假设FraR转录因子是一种抑制因子。因此，防止其从fra操纵子启动子释放也具有治疗意义。我们建议确定FraR的天然诱导剂，并确定FraR在Fra操纵子中的DNA结合位点。在第二个目标中，我们计划采用宏基因组学，在F-Asn存在下的选择性生长和生物信息学的组合来测试以下想法：在健康的肠道社区中，有选择的微生物群成员利用F-Asn并防止沙门氏菌获得这种营养素。最后，我们希望我们对沙门氏菌中F-Asn利用的酶学和调节以及可能的竞争肠道微生物的研究结果，能够为我们设计新的益生菌的努力提供信息，这些益生菌可以降低小鼠沙门氏菌感染的严重程度和持续时间。总的来说，我们的努力将导致更好地了解沙门氏菌在发炎肠道中的生长和新的治疗方法。

 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.