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

革兰氏阳性细菌金黄色葡萄球菌在世界各地引起显著的发病率和死亡率。尽管S。金黄色葡萄球菌无症状地在30%的人口中定植，但它仍然可以引起各种疾病，从败血症到浅表皮肤感染。由于这种疾病已经在医院和社区获得了对各种抗生素的耐药性，其严重程度正在惊人地上升。目标1：检查细胞壁锚定蛋白的个体作用，并评估它们在不同环境条件下对补体的保护作用，旨在模拟体内感染。在这里，学生将创建关键细胞壁蛋白的同基因突变体和双突变体使用等位基因交换基因缺失或pTnT产生来自内布拉斯加州转座子库的双和三重突变体。在这里，我们将测试细菌在正常血清和IgG/IgM耗尽的血清中孵育时，哪些细菌因素在限制补体沉积中是必不可少的。在不同条件下生长的细菌将说明S.金黄色葡萄球菌限制补体活性。进一步的分析将研究可溶性补体抑制因子H（FH）是否被S。金黄色葡萄球菌，实验将确定什么是S。金黄色葡萄球菌蛋白介导这种募集。目前尚不清楚S.金黄色葡萄球菌，这些机制的阐明提供了治疗干预的目标。目标二：学生将使用启动子报告质粒和创新的转录组学分析，采用GFP报告载体和高分辨率RNA测序来确定实验室和体内条件下的差异基因表达。 Laabei实验室以前的工作表明，双组分系统（TCS），GraRS，在调节补体evasins的表达中是必不可少的;然而，确切地说，这种GraRS携带的补体evasins是未知的。使用启动子报告和RNAseq，我们将确定和验证由GraRS控制的补体evasins调节子。目的3探讨补体evasins在链球菌存活中的作用。金黄色葡萄球菌学生将开发一个人类血液挑战模型，并确定1）血液存活所需的补体evasin和2）通过使用一组化学和抗体抑制剂来抑制补体和抗体介导的吞噬作用，抑制血小板活化和补体炎症片段的产生，哪些宿主因子对血液杀伤至关重要。这些数据将揭示抑制和增强血液介导的杀伤的基本靶点，并鉴定介导S。金黄色葡萄球菌从血液中消除。最后，学生将在一组临床相关的，遗传多样性的基因组测序的S。金黄色葡萄球菌分离株。目标4将采用功能基因组学方法，结合基因型和表型，使全基因组关联研究（GWAS），以确定与增加或减少免疫逃避相关的遗传特征。这些基因特征将在实验室中进行进一步测试和功能确认，揭示与补体逃避相关的新基因和突变。利用这些数据，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株的免疫逃避性，这是了解致病性和改善疾病管理的重要一步。

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.