16S rRNA seq-identified Corynebacterium promotes pyroptosis to aggravate diabetic foot ulcer.

ABSTRACT

BACKGROUND: Diabetic foot ulcer (DFU) is one of the main chronic complications caused by diabetes, leading to amputation in severe cases. Bacterial infection affects the wound healing in DFU. METHODS: DFU patients who met the criteria were selected, and the clinical data were recorded in detail. The pus exudate from the patient's foot wound and venous blood were collected for biochemical analysis. The distribution of bacterial flora in pus exudates of patients was analyzed by 16S rRNA sequencing, and the correlation between DFU and pathogenic variables, pyroptosis and immunity was analyzed by statistical analysis. Then, the effects of key bacteria on the inflammation, proliferation, apoptosis, and pyroptosis of polymorphonuclear leukocytes were investigated by ELISA, CCK-8, flow cytometry, RT-qPCR and western blot. RESULTS: Clinical data analysis showed that Wagner score was positively correlated with the level of inflammatory factors, and there was high CD3+, CD4+, and low CD8+ levels in DFU patients with high Wagner score. Through alpha, beta diversity analysis and species composition analysis, Corynebacterium accounted for a large proportion in DFU. Logistics regression model and Person correlation analysis demonstrated that mixed bacterial infections could aggravate foot ulcer, and the number of bacteria was closely related to inflammatory factors PCT, PRT, immune cells CD8+, and pyroptosis-related proteins GSDMD and NLRP3. Through in vitro experiments, Corynebacterium inhibited cell proliferation, promoted inflammation (TNF-α, PCT, CRP), apoptosis and pyroptosis (IL-1ß, LDH, IL-18, GSDMD, NLRP3, and caspase-3). CONCLUSION: Mixed bacterial infections exacerbate DFU progression with a high predominance of Corynebacterium, and Corynebacterium promotes inflammation, apoptosis and pyroptosis to inhibit DFU healing.