Characterisation of a novel protein toxin family secreted by the animal and human pathogen Staphylococcus aureus
动物和人类病原体金黄色葡萄球菌分泌的新型蛋白质毒素家族的表征
基本信息
- 批准号:2753148
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:英国
- 项目类别:Studentship
- 财政年份:2022
- 资助国家:英国
- 起止时间:2022 至 无数据
- 项目状态:未结题
- 来源:
- 关键词:
项目摘要
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.
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,导致产奶量显著减少。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药耐药基因亚型可以追溯到牛。为了引起疾病S。金黄色葡萄球菌必须首先在宿主中定殖。要做到这一点,它必须有效地与居民微生物群竞争,以建立一个利基。PI的实验室已经证明S。金黄色葡萄球菌使用其VII型蛋白分泌系统(T7 SS)分泌核酸酶和靶向其他细菌的膜去极化毒素。S.金黄色葡萄球菌通过共同产生中和毒性活性的免疫蛋白而免受其自身毒素的作用。到目前为止,所有的S。金黄色葡萄球菌T7 SS分为三个蛋白家族:WXG 100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鉴定了第四个底物蛋白家族,这些蛋白质尚未在文献中描述。S.金黄色葡萄球菌编码这些新底物中的两种,但在许多其他革兰氏阳性细菌中发现了该蛋白质家族。本项目的目的是对这些新的S.金黄色葡萄球菌T7 SS,并通过分子分析确定其毒性活性。使用的方法:为了测试毒性活性,两种底物都将在E. coli和S. aureus在可调控的启动子下。靶向序列将被添加到毒素结构域以将它们引导到膜的细胞外侧(以确定它们的靶标是否驻留在该隔室中)。免疫蛋白通常编码在其毒素伴侣附近3。每种毒素附近的基因将被测试,以确定它们是否能保护免受毒性活动的影响。毒素和免疫蛋白之间的直接相互作用将使用细菌双杂交和免疫共沉淀方法来检测。建模方法将用于预测毒素的结构/功能,并将设计和测试候选活性位点的替代毒性试验。毒素和免疫蛋白将分别和以复合物的形式过量产生(在缺乏免疫伴侣的情况下,可能需要使用无活性的毒素变体进行表达)。纯化的蛋白质将用于结构分析。将进行一系列表型实验以鉴定每种毒素的细胞靶标。这将得到生物化学实验的支持,以证明体外生物活性。将构建候选毒素和免疫蛋白的单个染色体缺失,用于细菌竞争实验。将使用PI实验室建立的新型斑马鱼胚胎定殖模型来评估细菌间竞争。后脑提供了一个无菌室,其中可以共接种两种细菌菌株。T7 SS在这些体内条件下具有高度活性,并且在9小时后观察到靶菌株的杀伤,并且可以使用细菌计数进行定量。该模型将用于评估每种毒素在杀死(i)密切相关的S.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(例如,S.溶血性链球菌S.表皮葡萄球菌和表皮葡萄球菌。人)(iii)其他乳腺相关物种,如拟杆菌和双歧杆菌。这里提议的工作与BBSRC在农业和粮食安全方面的战略优先事项密切相关。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,如动物健康和福利。
项目成果
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其他文献
吉治仁志 他: "トランスジェニックマウスによるTIMP-1の線維化促進機序"最新医学. 55. 1781-1787 (2000)
Hitoshi Yoshiji 等:“转基因小鼠中 TIMP-1 的促纤维化机制”现代医学 55. 1781-1787 (2000)。
- DOI:
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LiDAR Implementations for Autonomous Vehicle Applications
- DOI:
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2021 - 期刊:
- 影响因子:0
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吉治仁志 他: "イラスト医学&サイエンスシリーズ血管の分子医学"羊土社(渋谷正史編). 125 (2000)
Hitoshi Yoshiji 等人:“血管医学与科学系列分子医学图解”Yodosha(涉谷正志编辑)125(2000)。
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Effect of manidipine hydrochloride,a calcium antagonist,on isoproterenol-induced left ventricular hypertrophy: "Yoshiyama,M.,Takeuchi,K.,Kim,S.,Hanatani,A.,Omura,T.,Toda,I.,Akioka,K.,Teragaki,M.,Iwao,H.and Yoshikawa,J." Jpn Circ J. 62(1). 47-52 (1998)
钙拮抗剂盐酸马尼地平对异丙肾上腺素引起的左心室肥厚的影响:“Yoshiyama,M.,Takeuchi,K.,Kim,S.,Hanatani,A.,Omura,T.,Toda,I.,Akioka,
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