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

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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.