Delineating the mechanisms utilised by Staphylococcus aureus to survive intracellularly within phagocytes

Abstract

Staphylococcus aureus (S. aureus) is a major human pathogen in addition to being an important part of the human microbiome. The bacterium has evolved a large array of immune evasion mechanisms to facilitate its persistence including the ability to survive intracellularly within phagocytes. The intracellular lifestyle of S. aureus and the mechanisms that underpin it are poorly understood and so this thesis aimed to understand the immunosuppressive features that enable intracellular persistence within phagocytes. Total RNA was isolated from human neutrophils (hPMN) and murine bone derived macrophages harboring intracellular S aureus for multiplex gene expression analysis. Additionally, neutrophil RNA was assessed for miRNA expression to understand the roles of miRNA in governing neutrophil gene expression during intracellular survival of S. aureus. Gene expression analysis of S. aureus infected hPMN revealed upregulation of the adora2a receptor (A2aR) which was found to suppress neutrophil function. A2aR activation led to metabolic reprogramming of infected hPMN by enhancing glycolysis at the cost of the pentose phosphate pathway limiting NADPH and significantly reducing ROS production. Additionally, A2aR activation led to decreased NETosis and proinflammatory cytokine production but could be rescued using an A2aR antagonist. hPMN miRNA was also found to impact intracellular survival with a subset of miRNA significantly downregulated in hPMN harboring intracellular S. aureus compared to uninfected neutrophils. Further interrogation of these miRNAs with predictive analysis software revealed TGF-β signaling was likely upregulated in hPMN harboring intracellular S. aureus. Intervention with the TGF-β and activin signaling antagonist led to significant reductions in intracellular burden. Intracellular survival of S. aureus within macrophages was also assessed with a view to identify pathways which may facilitate S. aureus nasal colonization given that S. aureus can drive an immunosuppressive environment for colonization. Gene expression analysis revealed a strain dependent induction of type 1 interferons (IFN-I) which led to the induction of the proapoptotic factors trail and daxx. S. aureus induced IFN-I led to macrophage apoptosis both in vitro and in vivo during nasal colonization. Loss of IFN-I ablated apoptosis in vitro and significantly decreased nasal colonization whilst inhibition of caspase-3 led to similar reductions in nasal colonization. In all, this thesis highlighted the adept and pathogenic ability of intracellular S. aureus to evade phagocytic killing whilst also highlighting the ability of S. aureus to reshape the nasal cavity for its commensal lifestyle. The work laid out in this project substantially broadens our understanding of intracellular S. aureus and highlights several opportunities for novel host directed therapeutic.