Characterisation of a novel protein toxin family secreted by the animal and human pathogen Staphylococcus aureus

革兰氏阳性细菌，金黄色葡萄球菌，是一种哺乳动物病原体。它是皮肤和软组织感染的主要原因，也是牛乳腺炎的常见原因，导致牛奶产量显著减少。这种生物可以在宿主物种之间跳跃，至少有两种在人类中流行的多重耐药遗传亚型可以追溯到牛。为了引起疾病，金黄色葡萄球菌必须首先在宿主体内定殖。要做到这一点，它必须有效地与常驻微生物群竞争，以建立一个生态位。PI的实验室已经证明，金黄色葡萄球菌使用其VII型蛋白分泌系统（T7SS）分泌核酸酶和针对其他细菌的膜去极化毒素。金黄色葡萄球菌通过共同产生免疫蛋白来中和毒性活性，从而免受自身毒素的作用。迄今为止，金黄色葡萄球菌T7SS的所有特征底物都属于三个蛋白家族：WXG100蛋白、YeeF结构域蛋白和LXG结构域蛋白。最近，PI通过蛋白质组学和基因组学分析确定了第四家族的底物蛋白，这在文献中尚未描述。金黄色葡萄球菌编码其中两种新型底物，但在许多其他革兰氏阳性细菌中发现了这种蛋白质家族。该项目的目的是表征金黄色葡萄球菌T7SS的这些新底物，并通过分子分析确定其毒性活性。使用方法：为了测试毒性活性，这两种底物将在大肠杆菌和金黄色葡萄球菌中在可调节的启动子下产生。靶向序列将被添加到毒素结构域，以引导它们到膜的细胞外侧（以确定它们的目标是否位于这个隔室）。免疫蛋白通常与它们的毒素伴侣相邻编码。每一种毒素的邻近基因都将被测试，看它们是否能防止毒性活动。毒素与免疫蛋白之间的直接相互作用将采用细菌双杂交和共免疫沉淀方法进行检测。建模方法将用于预测毒素的结构/功能，候选活性位点替代将被设计并在毒性分析中进行测试。毒素和免疫蛋白将分别和复合过量产生（在没有免疫伴侣的情况下，可能需要使用无活性的毒素变体进行表达）。纯化后的蛋白质将用于结构分析。将进行一系列表型实验，以确定每种毒素的细胞目标。这将得到生化实验的支持，以证明体外的生物活性。候选毒素和免疫蛋白的单个染色体缺失将被构建用于细菌竞争实验。细菌间竞争将使用在PI实验室建立的新型斑马鱼胚胎定植模型进行评估。后脑提供了一个无菌室，在那里两种细菌菌株可以共同接种。T7SS在这些体内条件下具有高度活性，在9小时后观察到目标菌株的杀死，并可以通过细菌计数进行定量。该模型将用于评估每种毒素在杀死(i)密切相关的金黄色葡萄球菌菌株（ii）与牛乳腺相关的更多样化的葡萄球菌（如溶血葡萄球菌、表皮葡萄球菌和人型葡萄球菌）（iii）其他与乳腺相关的物种（如拟杆菌和双歧杆菌）中的作用。这里提出的工作与BBSRC在农业和粮食安全方面的战略重点密切相关。这一优先事项的一个关键方面是支持对粮食安全和食品安全具有深远影响的领域的研究，例如动物健康和福利。

The Gram-positive bacterium, Staphylococcus aureus, is a mammalian pathogen. It is a major cause of skin and soft tissue infections, and a frequent cause of bovine mastitis, resulting in a significant reduction in milk production. The organism can jump between host species, and at least two multi-drug resistant genetic subtypes that are endemic in people have been traced back to cattle. In order to cause disease S. aureus must first colonise the host. To do this it must effectively compete with the resident microbiota to establish a niche. The PI's lab has demonstrated that S. aureus uses its Type VII protein secretion system (T7SS) to secrete nuclease and membrane-depolarizing toxins that target other bacteria. S. aureus is protected from the action of its own toxins by the co-production of immunity proteins that neutralize toxic activity. To date, all characterised substrates of the S. aureus T7SS fall into three protein families: the WXG100 proteins, the YeeF domain proteins and the LXG domain proteins. Very recently the PI has identified a fourth family of substrate proteins through proteomic and genomic analysis that have not yet been described in the literature. S. aureus encodes two of these novel substrates, but the protein family is found across many other Gram-positive bacteria. The aim of this project is to characterise these new substrates of the S. aureus T7SS and to define their toxic activities through molecular analysis. Approaches to be used: To test for toxic activity, both substrates will be produced in E. coli and in S. aureus under a regulatable promoter. Targeting sequences will be added to the toxin domains to direct them to the extracellular side of the membrane (to determine whether their targets reside in this compartment). Immunity proteins are usually encoded adjacently to their toxin partners3. Genes in the immediate neighbourhood of each toxin will be tested to see whether they protect from toxic activity. Direct interaction between toxins and immunity proteins will be detected using bacterial two hybrid and co-immunoprecipitation approaches. Modelling approaches will be used to predict structure/function of toxins, and candidate active site substitutions will be designed and tested in toxicity assays. Toxins and immunity proteins will be overproduced separately and in complex (it may be necessary to use an inactive toxin variant for expression in the absence of an immunity partner). Purified proteins will be used for structural analysis. A range of phenotypic experiments will be undertaken to identify the cellular target each toxin. This will be supported by biochemical experiments to demonstrate biological activities in vitro.Individual chromosomal deletions of candidate toxins and immunity proteins will be constructed for use in bacterial competition experiments. Interbacterial competition will be assessed using a novel zebra fish embryo colonisation model that has been established in the PI's laboratory. The hindbrain provides a sterile compartment where two bacterial strains can be co-inoculated. The T7SS is highly active under these in vivo conditions and killing of target strains is observed after 9 hours and can be quantitated using bacterial counts. This model will be used to assess the roles of each toxin in the killing of (i) closely related S. aureus strains (ii) more diverse Staphylococci associated with the bovine mammary gland (e.g. S. haemolyticus, S. epidermidis and S. hominis) (iii) other mammary gland-associated species such as Bacteroides and Bifidobacteria. The work proposed here maps closely to the BBSRC's strategic priority in agriculture and food security. A key aspect of this priority is to support research in areas that have profound implications for food security and food safety such as animal health and welfare.