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Title: Identifying novel immune modulating factors in a genome-wide Staphylococcus aureus screen in human neutrophils
Author: Yang, Dingyi
ISNI:       0000 0004 6500 3800
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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The multi-antibiotic resistant Staphylococcus aureus continues to be a worldwide clinical burden and there is an urgent need for novel therapeutic strategies. Neutrophils are essential during the innate immune response to S. aureus, yet this pathogen uses multiple evasion mechanisms to survive or even replicate within neutrophils. In addition, S. aureus induces rapid and profound neutrophil cell death, which further subverts the immune response. The aim of this project was to identify novel immune modulating genes of S. aureus through the screening of a genome-wide S. aureus mutant library of 1,920 strains in a neutrophil cell death assay. The mutant library was constructed by transposon insertion in the clinically relevant community acquired methicillin-resistant S. aureus (USA300) background. Individual S. aureus strains were co-incubated with primary human neutrophils isolated from healthy subjects, at an MOI of 10 for 3 hours before ToPro-3 staining and assessment of cell loss via flow cytometry. A number of internal controls with known pro-death functions including lukGH, agrA and saeS were among the 34 strains that resulted in attenuated cell death. Four gene mutations not previously associated with cell death: purB, lspA, clpP and pfo were also identified and subsequently verified by genetic transduction. The clpP and lspA were further validated by gene complementation. The purine synthesis pathway (purB) was found to be important for bacterial replication and pathogenesis in a zebrafish embryo model, yet this in vivo phenotype was proved to be neutrophil-independent. Lack of lspA led to significantly reduced phagocytosis, indicating the necessity of lipoproteins in bacterial recognition and regulation of neutrophil lysis. Pyruvate metabolism (pfo) was required for neutrophil cell lysis in vitro. Yet no attenuated phenotype of pfo or lspA was found in vivo, possibly due to host-dependent effects. The clpP mutant failed to grow or induce host mortality in wild type zebrafish embryos, but virulence was restored in a phagocyte-depleted model, suggesting the importance of clpP in bacterial survival and pathogenesis, possibly via Clp ATPase regulated post-phagocytosis stress tolerance and clpP modulated expression of secreted toxins. This thesis identified novel gene functions and suggests possible mechanisms of S. aureus induced neutrophil cell lysis, which may aid the design of future antibioticindependent therapeutic strategies to restore failures of innate immunity.
Supervisor: Prince, Lynne ; Foster, Simon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available