Escaping host immunity: Characterising immune evasion mechanisms employed by the bacterial pathogen Staphylococcus aureus.

革兰氏阳性菌金黄色葡萄球菌在世界各地造成了严重的发病率和死亡率。即使金黄色葡萄球菌无症状地在30%的人口中定居，它仍然可以引起各种疾病，从败血症到浅表皮肤感染。由于这种疾病在医院和社区对多种抗生素产生了抗药性，其严重性正在令人担忧地上升。目的1：研究细胞壁锚定蛋白在不同环境条件下对补体的保护作用，以模拟体内感染。在这里，学生将创建关键细胞壁蛋白的等基因突变和使用等位基因交换基因缺失或pTnT的双重突变，以产生来自内布拉斯加州转座子文库的双重和三重突变。在这里，我们将测试哪些细菌因素在限制补体沉积方面是必不可少的，当细菌在正常血清和IgG/IgM耗尽的血清中孵育时。这些细菌在不同的条件下生长，将说明金黄色葡萄球菌限制补体活性的能力。进一步的分析将调查金黄色葡萄球菌是否招募了可溶性补体抑制因子H(FH)，并将通过实验确定哪些金黄色葡萄球菌蛋白介导了这种招募。目前在金黄色葡萄球菌中，分泌型免疫逃逸分子是如何被调控的尚不清楚，阐明这些机制为治疗干预提供了靶点。目标2：学生将使用启动子-报告质粒和创新的转录分析，使用GFP报告载体和高分辨率RNA测序来确定在实验室和类似体内条件下的差异基因表达。拉贝实验室以前的工作表明，双组分系统(TCS)GRARS在调节补体Easins的表达方面是必不可少的；然而，这种GRARS到底含有哪种补体Easins尚不清楚。利用启动子Reports和RNAseq，我们将确定并验证GraRS控制的补体Easins调节子。目的3探讨补体伊维菌素在人血中金黄色葡萄球菌存活中的作用。学生将开发一个人类血液挑战模型，并确定1)血液生存需要哪些补体伊瓦菌素(S)，以及2)通过使用一组化学和抗体抑制剂，使补体和抗体介导的吞噬功能失活，抑制血小板激活和补体炎症片段的产生，哪些宿主因素对血液杀戮是必不可少的。这些数据将揭示抑制和增强血液介导的杀戮的基本靶点，并确定介导金黄色葡萄球菌从血液中消除的宿主成分。最后，学生将检查一组临床相关的、基因多样化的金黄色葡萄球菌分离株的补体逃逸情况。目标4将使用一种功能基因组学方法，将基因型和表型结合起来，使全基因组关联研究(Gwas)能够识别与免疫逃避增加或减少相关的遗传特征。基因签名将在实验室进行进一步的测试和功能确认，揭示与补体逃避有关的新基因和突变。使用这些数据，学生将优化机器学习方法，直接从细菌基因组序列预测分离物的免疫逃避能力，这是了解致病性和改善疾病管理的关键一步。

The gram-positive bacterium Staphylococcus aureus causes significant morbidity and mortality around the world. Even though S. aureus asymptomatically colonises 30% of the population, it can still cause various illnesses, from sepsis to superficial skin infections. Because this disease has acquired resistance to a wide range of antibiotics in hospitals and communities, its severity is rising alarmingly. Objective 1: Examine the individual role of cell wall anchored proteins and assess their contribution to protection against complement under different environmental conditions aimed at mimicking in vivo infection. Here, the student will create isogenic mutants of crucial cell wall proteins and double mutants using allelic exchange gene deletion or pTnT to generate double and triple mutants derived from the Nebraska Transposon library. Here, we will test what bacterial factors are essential in limiting complement deposition when bacteria are incubated in normal serum and IgG/IgM-depleted serum. The bacteria, growing under different conditions, will illustrate the ability of S. aureus to limit complement activity. Further analysis will investigate whether the soluble complement inhibitor Factor H (FH) is recruited by S. aureus, and experiments will determine what S. aureus proteins mediate this recruitment. How secreted immune evasion molecules are regulated is currently unclear in S. aureus, and elucidation of these mechanisms offers targets for therapeutic intervention. Objective 2: The student will use promoter-reporter plasmids and innovative transcriptomic analysis, employing GFP reporter vectors and high-resolution RNA sequencing to determine differential gene expression under lab and in vivo-like conditions. Previous work of the Laabei lab has shown that the two-component system (TCS), GraRS, is essential in regulating the expression of complement evasins; however, exactly which complement evasins this GraRS harbours is unknown. Using promoter reporters and RNAseq, we will determine and validate the complement evasins regulon controlled by GraRS. Objective 3 will investigate the role of complement evasins in the survival of S. aureus in human blood. The student will develop a human blood challenge model and determine 1) what complement evasin(s) are required for blood survival and 2) what host factors are essential for blood killing by using a panel of chemical and antibody inhibitors that inactive complement and antibody-mediated phagocytosis, inhibit platelet activation and the generation of complement inflammatory fragments. This data will reveal essential targets to inhibit and enhance blood-mediated killing and identify host components that mediate S. aureus elimination from the blood. Lastly, the student will examine complement evasion in a cohort of clinically relevant, genetically diverse genome-sequenced S. aureus isolates. Objective 4 will employ a functional genomics approach, combining genotype and phenotype, enabling genome-wide association studies (GWAS) to identify genetic signatures associated with increased or decreased immune evasiveness. The genetic signatures will be further tested and functionally confirmed in the lab, revealing novel genes and mutations linked to complement evasion. Using this data, the student will optimise machine learning methodology to predict the immune evasiveness of an isolate directly from the bacterial genome sequence, an essential step towards understanding pathogenicity and improving disease management.