Abstract
Staphylococcus aureus is a commensal organism in approximately 30% of the human population and colonization is a significant risk factor for invasive infection. As a result of this, there is a great need to better understand how S. aureus overcomes human immunity. Neutrophils are essential during the innate immune response to S. aureus, yet this microorganism uses multiple evasion strategies to avoid killing by these immune cells, perhaps the most catastrophic of which is the rapid induction of neutrophil cell death. The aim of this study was to better understand the mechanisms underpinning S. aureus-induced neutrophil lysis, and how this contributes to pathogenesis in a whole organism model of infection. To do this we screened the genome-wide Nebraska Transposon Mutant Library (NTML) in the community acquired methicillin resistant S. aureus strain, USA300, for decreased ability to induce neutrophil cell lysis. Out of 1,920 S. aureus mutants, a number of known regulators of cell lysis (including the master regulators accessory gene regulator A, agrA and Staphylococcus exoprotein expression protein S, saeS) were identified in this blinded screen, providing validity to the experimental system. Three gene mutations not previously associated with cell death: purB, lspA, and clpP were found to be significantly attenuated in their ability to induce neutrophil lysis. These phenotypes were verified by genetic transductants and complemented strains. purB and clpP were subsequently found to be necessary for bacterial replication and pathogenesis in a zebrafish embryo infection model. The virulence of the clpP mutant was restored in a neutrophil-depleted zebrafish model, suggesting the importance of ClpP in mechanisms underpinning neutrophil immunity to S. aureus. In conclusion, our work identifies genetic components underpinning S. aureus pathogenesis, and may provide insight into how this commensal organism breaches innate immune barriers during infection.
Highlights
Staphylococcus aureus has long been recognized as a highly adaptive and dangerous human pathogen, yet this microorganism colonizes the nose of ∼30% of the human population without any ill effects [1]
While this is of great interest, and may reflect a loss of a negative regulatory mechanism of cell death, we did not further pursue these mutants, since our objective was to identify genes that played a positive role in the induction of neutrophil lysis
Preserving neutrophil function during infection by preventing S. aureus-induced cell death is an attractive therapeutic strategy and here we describe three genes with previously unidentified roles in neutrophil cell death
Summary
Staphylococcus aureus has long been recognized as a highly adaptive and dangerous human pathogen, yet this microorganism colonizes the nose of ∼30% of the human population without any ill effects [1]. Virulent methicillin resistant S. aureus (MRSA) strains can be carried asymptomatically by healthy individuals [2]. Interventions such as hospitalization or episodes of immunosuppression can result in invasive S. aureus infection, which can manifest in multiple forms from superficial skin abscesses to necrotising pneumonia or life-threatening bacteraemia. Neutrophils are a critical defense in controlling colonization and active infection with S. aureus [5, 6] This microorganism uses multiple evasion strategies to avoid killing by these innate immune cells [reviewed in [7]]. Perhaps the most catastrophic of these strategies is the induction of neutrophil cell death This eradicates a critical element of the early immune response, and results in inflammation and tissue damage which intensifies disease. The increased virulence of CAMRSA has been attributed in part to its resistance to neutrophilmediated killing, including via the induction of neutrophil lysis [14]
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