Genetically Modifying Lactobacillus to Alter Gut Inflammation and Pathogens

本课题主要研究了乳酸菌表达1型干扰素的特性，并研究了对照和转基因乳酸菌对葡聚糖硫酸钠诱导的肠道炎症的影响。我们成功地证明了表达干扰素的细菌而不是对照细菌触发了STAT1的快速磷酸化，这一效应需要细菌与巨噬细胞之间的接触。此外，这种作用依赖于干扰素-β受体，而不需要Toll受体2或9。乳杆菌表达干扰素基因还诱导了原代小鼠树突状细胞和小鼠肠道上皮细胞株中STAT1的快速磷酸化。我们还证明了转基因细菌可以直接分泌干扰素-β。目前关于细菌对DSS诱导的肠道炎症的影响的研究取得了意想不到的结果。我们观察到，给予产生干扰素-β的乳杆菌实际上加重了DSS诱导的结肠炎，我们目前的数据支持这样一个模型，即干扰素-β的表达干扰肠道树突状细胞的成熟，导致更有利于炎症的环境。作为该项目的第二部分，我们开发了一种检测小鼠诺沃克病毒的聚合酶链式反应方法，并已确定在感染后至少10-12天内可以在粪便颗粒中检测到诺沃克病毒。我们现在正在测试注射乳酸菌是否可以改变感染的时间进程。作为该项目的新部分，我们发现乳杆菌似乎具有趋化活性，我们正在对培养上清液进行纯化，试图鉴定其活性成分。

This project has focused on characterizing the expression of the Type 1 interferon (IFN) by Lactobacillus and determining the consequences of administering control and transgenic Lactobacillus on dextran sodium sulfate induced gut inflammation. We successfully demonstrated that bacteria expressing the interferon but not the control bacteria, triggered rapid STAT1 phosphorylation and that this effect required contact between the bacteria and the macrophages. Furthermore, this effect was dependent on the interferon-beta receptor but did not require toll receptors 2 or 9. The expression of the interferon gene by the Lactobacillus also induced rapid STAT1 phosphorylation in primary murine dendritic cells as well as a mouse gut epithelial cell line. We also demonstrated direct secretion of IFN-beta by the transgenic bacteria. Current studies on the effects of the bacteria on DSS induced gut inflammation have yielded unexpected results. We have observed that administration of IFN-beta producing Lactobacillus actually worsens DSS induced colitis and our current data supports a model where the IFN-beta expression interferes with gut dendritic cell maturation, resulting in a more pro-inflammatory environment. As a second part of this project, we have developed a PCR assay for murine norovirus and have determined that norovirus can be detected in fecal pellets for at least 10-12 days following infection. We are now testing if administration of the Lactobacillus can alter the time course of infection. As a new part of this project, we have found that the Lactobacillus appear to secrete a chemotactic activity and we are in the process of purifying culture supernatant in an attempt to identify the active component.