Salmonella, colonization resistance, and fructose-asparagine

 说明(申请人提供)：肠沙门氏菌(鼠伤寒沙门氏菌)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，因为沙门氏菌在肠道中有太多的营养可用。然而，我们发现，在发炎的肠道中，沙门氏菌的生长严重依赖于一种单一的营养物质--果糖-天冬酰胺(F-ASN)，这种营养物质在人类食物中含量很高。不能获得F-ASN的突变株严重减弱，这表明F-ASN是沙门氏菌在炎症过程中利用的主要营养物质。目前还没有其他生物合成或利用这种化合物的报道，尽管我们怀疑一些其他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中明显缺乏F-ASN利用途径，这表明沙门氏菌有一个特异和有效的治疗靶点。只有当沙门氏菌能够引发炎症并使用四硫酸盐作为厌氧呼吸的末端电子受体时，编码F-ASN利用的基因位fra才能提供优势(fra表型分别在沙门氏菌SPI1-SPI2-或ttrA突变体中丢失)。我们假设，如果沙门氏菌可以引发炎症(或进入已经轻微炎症的肠道)，它可以在F-ASN分解代谢过程中开始四硫键呼吸，从而超过正常的微生物区系，后者受到炎症和只发酵(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建立基础，以阻止沙门氏菌获得F-ASN。在我们的第一个特定目标中，我们将研究天冬酰胺酶(FRAE)、激酶(FRAD)和脱氨酶(FRAB)在F-ASN利用中的作用。通过对这些Fra酶催化的个别反应进行生化表征和高通量分析的发展，我们预计将促进未来的筛选，以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，阻止其从fra操纵子启动子中释放也具有治疗意义。我们建议确定FraR的自然诱导剂，并确定FraR在Fra操纵子中的DNA结合部位。在第二个目标中，我们计划结合元基因组学、在F-ASN存在下的选择性生长和生物信息学来测试在健康的肠道社区中存在利用F-ASN的微生物区系的选定成员并防止沙门氏菌获得这种营养的想法。最后，我们期待我们在沙门氏菌中对F-ASN利用的酶学和调节方面的发现，以及可能的竞争肠道微生物，为我们设计能够降低沙门氏菌感染的严重程度和持续时间的新的益生菌的努力提供参考。总体而言，我们的努力将使我们更好地了解沙门氏菌在炎症肠道中的生长情况，并找到新的治疗方法。

 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.