Characterisation of the pathogenesis and immunogenicity of two novel attenuated mutants of Salmonella Typhimurium

肠道沙门氏菌会在许多不同的动物身上引起许多疾病。此前，我们使用自己的新技术测试了一种名为鼠伤寒沙门氏菌的特定类型肠杆菌中的每个基因，以确定该基因是否是细菌在小鼠感染模型中致病所必需的。在确定了细菌中几乎每个基因对毒力的贡献程度后，我们现在需要跟踪一些更有趣的基因，以找出它们在疾病过程中是如何工作的。我们还需要了解为什么缺乏上述基因的细菌突变不能导致完全感染，为什么它们不会引起沙门氏菌感染所常见的炎症，可能最重要的是，突变细菌在多大程度上以及如何作为活疫苗发挥作用。在这个项目中，我们建议表征ATPA和trxA突变体。在初步研究中，我们已经表明，这些突变株在小鼠中的生长速度比正常沙门氏菌慢得多(称为高度减毒)，并且它们可以保护小鼠免受随后感染正常、剧毒的沙门氏菌的影响，即它们可以作为良好的疫苗。这两种突变在感染动物的脾中引起的炎症很少，甚至没有，因此在疫苗株中使用这些突变可能是减少不良反应的一种方法。在这项研究中，将使用微生物学、细胞生物学和免疫学技术来彻底描述疫苗减毒、无炎症和作为疫苗的能力的基础。ATPA基因存在于沙门氏菌的DNA中，与其他几个类似的ATP基因相邻，这些基因的突变株将被构建并测试其引起感染的能力。同样，沙门氏菌中还有其他trx基因参与trxA基因产物的功能，这些基因也将发生突变，并测试其对沙门氏菌感染能力的影响。为了试图了解变种人缺乏什么使他们不能引起严重感染，他们将接受一系列的测试和化验。除了其他因素外，ATP基因还涉及一种对酸胁迫的抗性，因此将对酸的抗性进行测试。Trx基因参与了沙门氏菌处理不同氧气水平的系统，因此将测试对不同类型氧气的抵抗力。这些突变体在正常小鼠和缺乏某些抗细菌感染机制的小鼠的巨噬细胞中存活的能力将被测试，以确定这些机制是否被细菌基因产物所对抗。将测试突变体诱导信号的能力，这些信号通常是在各种遗传类型的细胞和动物中启动炎症所需的，以研究不引发炎症的机制。最后，将测试抗体和免疫细胞反应，试图了解这些突变体是如何诱导对正常沙门氏菌感染的保护性免疫的。该项目将提供有关这些新的候选疫苗株的详细信息，以及关于侵袭性沙门氏菌病的减毒、毒力、炎症和保护性免疫机制的大量数据。

Salmonella enterica causes many diseases in many different animals. Previously we used our own novel technology to test every gene in a particular type of S. enterica called serovar Typhimurium, for whether or not that gene is required for the bacteria to cause disease in a mouse model of infection. Having identified the extent to which almost every gene in the bacterium contributes to virulence we now need to follow up some of the more interesting genes to find out how they work in the disease process. We also need to understand why mutants of the bacteria lacking the genes in question cannot cause full infection, why they do not cause the inflammation that is normally seen with Salmonella infections, and possibly most importantly, to what extent and how the mutant bacteria act as live vaccines. In this project we propose to characterise atpA and trxA mutants. In preliminary studies we have shown that these mutants grow much more slowly than normal salmonellae in mice (known as being highly attenuated), and that they protect mice against subsequent infection with normal, virulent salmonellae, i.e. they can act as good vaccines. Both of these mutants induce minimal to no inflammation in the spleens of infected animals, thus using these mutations in vaccine strains may be a way of reducing adverse reactions. In this study the basis for the attenuation, lack of inflammation and ability to act as a vaccine will be thoroughly characterised using microbiological, cell biological, and immunological techniques. The atpA gene exists in the DNA of salmonellae next to several other similar atp genes, and mutants in these other genes will be constructed and tested for the ability to cause infection. Similarly, there are other trx genes in salmonellae that contribute to the function of the trxA gene product, and these too will be mutated and the effect of this on the ability of salmonellae to cause infection tested. To try to understand what it is that the mutants lack that makes them unable to cause severe infection, they will be subjected to a range of tests and assays. The atp genes are involved in a form of resistance to acid stress, amongst other things, so resistance to acid will be tested. The trx genes are involved, amongst other things, in systems that salmonellae have for dealing with different levels of oxygen, and so resistance to different types of oxygen will be tested. The ability of the mutants to survive in particular cells called macrophages from normal mice and mice that lack certain mechanisms for fighting bacterial infection will be tested to see whether these mechanisms are being countered by the bacterial gene products. The ability of the mutants to induce signals that are normally required to start inflammation in cells and animals of various genetic types will be tested to investigate the mechanism by which inflammation is not induced. Finally, antibody and immune cell responses will be tested to try to understand what it is that these mutants are doing that induces protective immunity against normal Salmonella infection. This project will provide detailed information about these new candidate vaccine strains, as well as considerable data concerning the mechanisms of attenuation, virulence, inflammation and protective immunity in invasive salmonellosis.