Staphylococcus aureus is a leading cause of nosocomial infections worldwide, placing a significant burden on both patients and the Australian healthcare system. The host innate immune response is crucial for combating S. aureus and is spearheaded by neutrophils. Migration of neutrophils to the site of infection, a process called chemotaxis, is responsible for initiating an effective immune response. How S. aureus interferes with chemotactic processes remains to be fully elucidated. Factors like the chemotaxis inhibitory protein of S. aureus have been reported, however, a full repertoire of genes involved in neutrophil chemotaxis modulation is unknown.
Here, we have developed a high-throughput ex-vivo approach built upon the Boyden Chamber assay to identify novel Staphylococcal factors that affect neutrophil chemotaxis. S. aureus cells were grown and transferred to bottom wells of a transwell plate. Human neutrophils from buffy coat products were seeded into the top wells. Neutrophils migrated to the bottom wells were quantified using a chromogenic substrate specific for human neutrophil elastase and compared to the chemotaxis induced by a wild-type S. aureus strain, JE2. Using this method, we screened 1076 mutants from the Nebraska Transposon Mutant Library, and identified 100 mutants with chemoattractive properties and 61 mutants with chemorepulsion properties. All 161 candidates were then independently validated in a traditional Boyden Chamber assay, after which 2 novel candidates were found to significantly modulate neutrophil chemotaxis (P < 0.05, one-way repeated measures ANOVA), one as a chemoattractant and another as a chemorepellent. Both candidates will be mechanistically characterized through further in-vitro and ex-vivo phenotypic assays, as well as proteomic methods. This research potentially advances our understanding of virulence and immune evasion strategies that S. aureus employs to persist during infection, and may lead to the identification of potential vaccine and therapeutic targets.