Determining the prevalence of the PGA biosynthetic operon in the genus Staphylococcus and the investigation of a PGA capsule depolymerase as a novel t

葡萄球菌是一组重要的细菌，经常在人类和动物的皮肤上发现。然而，一些物种，如耐甲氧西林金黄色葡萄球菌（MRSA）可导致危及生命的感染。在某些情况下，其他被称为凝固酶阴性葡萄球菌（CoNS）的葡萄球菌也会引起严重感染，通常发生在医院，也发生在重要的牲畜（如牛）中。令人担忧的是，这些细菌对抗生素的耐药性越来越强，使得感染更难治疗。因此，研究治疗这些感染的新方法非常重要。当引起感染时，这些细菌使用保护性胶囊或“外套”来躲避人类免疫系统。此外，在动物模型中已经表明，胶囊对于定殖是必不可少的，突出了这种毒力因子的重要性。该项目旨在开发一种治疗方法，快速清除这种胶囊，使宿主免疫系统能够识别感染并清除入侵的细菌。这种方法将涉及使用胶囊从细菌表面去除保护性聚-γ-谷氨酸（PGA）胶囊，从病原体表面剥离保护性胶囊。我们已经拥有一种这样的酶，并希望将其开发为治疗CoNS感染的新疗法。该项目将通过满足以下三个关键里程碑/目标，使用转化方法来测试这种疗法的潜力：首先，将确定CoNS组中PGA生物合成操纵子的流行率和遗传多样性，以确定哪些成员代表这种囊膜剥离疗法的可行靶标。我们还将对来自不同来源（人类和动物）的几种CoNS分离株的基因组进行测序，以进一步确认负责产生胶囊的基因的流行率和遗传多样性，特别是在感染牲畜的物种中。其次，将通过研究解聚酶处理对巨噬细胞摄取和杀死CoNS的影响来测试这种方法在体外对鉴定的物种的功效。这将使用落射荧光显微镜和荧光激活细胞分选（FACS）的组合来研究。解聚酶治疗解决或预防致死性菌血症的能力将使用葡萄球菌感染的大蜡螟模型进行研究。该模型还将允许我们通过使用qPCR测量参与先天免疫应答的关键基因的转录应答来监测治疗组与未治疗组中的Galleria免疫应答。该项目的成功结果将产生一种新的疗法，可用于治疗CoNS引起的人类和牲畜感染。

Staphylococci are an important group of bacteria and are often found on the skin of humans and animals. However, some species, like methicillin resistant Staphylococcus aureus (MRSA) can cause life threatening infections. Under certain conditions, other staphylococcal species known as Coagulase-negative Staphylocci or CoNS can also cause serious infections, often in hospitals but also in important livestock animals such as cattle. Worryingly, these species are becoming increasingly resistant to antibiotics making infections harder to treat. Therefore, it is important to investigate new ways of treating these infections.When causing an infection, these bacteria use a protective capsule or "coat" to hide from the human immune system. Additionally, it has been shown in animal models that the capsule is essential for colonisation, highlighting the importance of this virulence factor. This project aims to develop a treatment that will rapidly remove this capsule, allowing the host immune system to recognize the infection and clear the invading bacteria. This approach will have the benefit of being active against antibiotic resistant strains of CoNS.This approach will involve the use of a capsule to remove the protective poly-gamma-glutamic acid (PGA) capsule from the surface of the bacterium strip away the protective capsule from the surface of the pathogen. We already have one such enzyme in our possession and hope develop this as a new therapy for the treatment of CoNS infections. The project will use a translational approach to test the potential of this therapy by meeting the following three key milestones/objectives:Firstly, the prevalence and genetic diversity of the PGA biosynthetic operon within the CoNS group will be determined to identify which members represent viable targets of this capsule-stripping therapy. We will also sequence the genomes of several CoNS isolates from various sources (human and animal) to further confirm the prevalence and genetic diversity of the genes responsible for producing the capsule, especially in species infecting livestock. Secondly, the efficacy of this approach will be tested against identified species in vitro by investigating the effect of depolymerase treatment on macrophage uptake and killing of CoNS. This will be investigated using a combination of by using a combination of epifluorescent microscopy and fluorescence-activated cell sorting (FACS).Finally, the capacity for depolymerase therapy to resolve or prevent lethal bacteraemia will be investigated using a Galleria mellonella model of Staphylococcal infection. This model will also allow us to monitor the Galleria immune response in treated versus untreated groups by measuring the transcriptional response of key genes involved in the innate immune response using qPCR. Successful outcomes from this project will result in a novel therapy which can be used for the treatment of both human and livestock infections caused by CoNS.