S. aureus strain-level diversity in diabetic wound neutrophil phenotypes
The wound microbiome is hypothesized to influence healing and contribute to severe complications in diabetic foot ulcers (DFU). Staphylococcus aureus is a prevalent member of the DFU microbiome, frequently detected by cultures and DNA sequencing-based methods in clinically infected and uninfected DFU. While S. aureus is clearly a pathogen in various contexts, its role in the chronic diabetic wound remains elusive. Using shotgun metagenomic sequencing, we previously showed that strain-level variation of S. aureus in the DFU microbiome was associated with healing and clinical outcomes. From these same clinically uninfected DFU (n=100), S. aureus isolates were cultured; whole genome sequencing and functional screens suggest that neutrophil evasion and survival functions are enriched in S. aureus isolated from DFU with poor outcomes. These preliminary data lead us to hypothesize that strain-level diversity of S. aureus in the DFU microbiome drives interactions with neutrophils that modulate diabetic wound healing and outcomes. To test this hypothesis, we will execute two aims: AIM 1 will investigate the role of S. aureus strain-level variation in mediating neutrophil interactions and their association with clinical DFU outcomes. Using S. aureus isolates from a longitudinal, prospective cohort of 100 clinically uninfected, neuropathic DFUs, we will perform screens that target 3 major aspects of neutrophil-S. aureus interactions using primary human neutrophils: chemotaxis, phagocytosis, and survival. AIM 2 will determine the in vivo contribution of S. aureus strain-level diversity on neutrophil phenotypes, dynamics, and healing in the murine diabetic wound. We will perform excisional wound healing assays in diabetic mice (Leprdb/db; db/db) and infect with S. aureus DFU clinical isolates, comparing healing and neutrophil phenotypes in vivo. Completing these aims will shed light on the significance of strain-level variation in the wound microbiome and its impact on host-microbial interaction mechanisms during diabetic wound healing. The results will provide a foundation for studies to identify and characterize strain-level mechanisms that allow neutrophil evasion, focusing on those with the most relevance in our studies. We will also identify novel neutrophil markers for further investigation and validation during diabetic wound healing.

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