Abstract
Staphylococcus aureus is a multi-drug resistant human pathogen responsible for significant morbidity and mortality worldwide. Although S. aureus colonises approximately 30% of the population asymptomatically, it has the capacity to cause a variety of diseases ranging from mild skin infections to life-threatening bacteraemia. To disarm and bypass the host immune system, S. aureus possesses an extensive repertoire of virulence factors. One crucial component of the host innate immune system is the complement system, which consists of a proteolytic cascade of human plasma proteins that recruit effectors to the site of infection, labels pathogens for efficient phagocytosis and assemble into a pore-forming complex to directly lyse cells. Among pathogens, S. aureus has an unusually large repertoire of complement disarming proteins, collectively termed complement evasins (CE). However, our understanding of this critical evasion strategy has been severely limited by lack of suitable assays.In this study, we develop a novel method to examine S. aureus-mediated complement inactivation. We modified a serum bactericidal assay employing complement susceptible E. coli incubated with normal human serum in the presence or absence of S. aureus supernatant. Subsequent analysis of 14 genetically diverse S. aureus strains revealed CE as a variable virulence trait. Using phage transduction and an allelic exchange vector pIMAY*, we generated a library of individual secreted CE mutants in S. aureus backgrounds JE2 and Newman. Application of the novel assay to study the CE mutants identified evasins SCIN and SSL7 as essential for successful complement evasion. We optimised our assay to include computational colony counting, permitting high-throughput screening of 134 closely related genome sequenced clinical isolates. Our results revealed significant variability in CE even within the same clonal group. Through functional genomics, we identified a correlation between the presence of prophages and the pathogens’ ability to evade complement. We have also discovered a moderate correlation between isolate CE and cytotoxicity, and have identified multiple global regulators that are essential for complement evasion, particularly sensors of cell-wall stress, in promoting evasin expression.
Finally, we created a novel antibody-independent C3 probe derived from the staphylococcal Sbi protein, specifically the Sbi-IV domain. Using this probe, we show that biotin-labelled Sbi-IV specifically interacts with deposited C3 products on the staphylococcal surface and thus can be used to measure complement fixation on wild-type cells expressing a full repertoire of immune evasion proteins, including immunoglobulin binding proteins, and reinforced our observation in the variability of CE phenotype when applied to screen a panel of genetically diverse S. aureus.
Overall, our findings improve our understanding of the dynamic host-pathogen interaction and provide valuable tools for studying the CE phenotype in greater detail.
| Date of Award | 11 Oct 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Maisem Laabei (Supervisor) & Jean Van Den Elsen (Supervisor) |