A Genome-Wide Screen Identifies Factors Involved in S. aureus-Induced Human Neutrophil Cell Death and Pathogenesis

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.

Further Insight into the Mechanism of Human PMN Lysis following Phagocytosis of Staphylococcus aureus

“…Interestingly, lukGH is upregulated in human blood and the promoter is preferentially activated following exposure to PMNs ( 58 , 59 ). Moreover, a genome-wide screen identified LukGH as a prominent effector of PMN lysis caused by USA300 ( 26 ). DuMont et al identified CD11b as the cell surface receptor for LukAB (also known as LukGH) and showed that cytolysis of host cells by this leukocidin occurs through binding of CD11b on the cell surface ( 59 ).…”

Screening transcriptional connections in Staphylococcus aureus using high-throughput transduction of bioluminescent reporter plasmids

“…This annotated array of ~2000 mutants of all non-essential genes in the S. aureus JE2 background is a rich resource to investigate staphylococcal physiology. In its 96-well format, the library has been the basis for several phenotypic screens exploring the effects of individual mutations on various aspects of staphylococcal biology, including, but not limited to, S. aureus pathogenesis [12,13], adaptation to and survival of the host environment and corresponding stresses [14,15], and bacterial ability to withstand antimicrobials [16][17][18][19].…”

Multi-modal imaging reveals dynamic interactions ofStaphylococcus aureuswithin human neutrophils

“…NADPH oxidase and myeloperoxidase rapidly generate superoxide and HOCl which have strong antimicrobial properties (7,8). Despite these antimicrobial strategies, neutrophils fail to eliminate internalised S. aureus completely (9)(10)(11). Visualising dynamic host-pathogen interactions on a individual subcellular level, as opposed to in fixed samples and on a cell population level, allows us to better understand how S. aureus circumvents the neutrophil response and ultimately reveal pathways to therapeutically target to promote immunity during S. aureus infections.…”