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-1β, 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.

Keywords: 16S sRNA sequencing; Bacterial infection; Corynebacterium; Diabetic foot ulcer; Immune.

Fig. 1

A Wagner classification of 24 clinical patients with diabetic foot disease. B ELISA was used to detect the levels of serum TNF-α, CRP and PCT. C ELISA was performed to detect the expression of CD3 + , CD4 + and CD8 + in serum. D The ratio of CD4 + /CD8 + and PCT/CRP was calculated. E RT-qPCR was used to detect the relative expression of GSDMD and NLRP3

Fig. 2

Alpha diversity analysis. A Sparse curve. B Abundance grade curve. The richness of species is reflected by the length of the curve on the horizontal axis. The wider the curve, the richer the species composition. The uniformity of species composition is reflected by the shape of the curve. The more flat the curve is, the higher the uniformity of species composition is. The smoother the curve is, the higher the species diversity of the sample is, and the rapid and steep decline of the curve indicates that the proportion of dominant bacteria in the sample is high and the diversity is low. C Species accumulation curve

Fig. 3

Beta diversity analysis. A Hierarchical cluster analysis. The shorter the branch length between samples is, the more similar the two samples are. B Petal plot of species difference analysis. The common species and unique species between different sample groups were analyzed. C Distance matrix and PCoA analysis chart. Each point in the figure represents a sample, and points of different colors indicate different groups. The percentage in the coordinate axis brackets represents the proportion of sample difference data (distance matrix) that the corresponding coordinate axis can explain. D Non-metric multidimensional scaling (NMDS) analysis

Fig. 4

A Analysis of taxonomic composition. B Species composition map of metabolic pathways. C Metabolic pathway statistics

Fig. 5

A CCK-8 was used to detect cell viability. B Apoptosis was detected by flow cytometry. C The levels of inflammatory factors TNF-α, PCT and CRP in cells were measured by ELISA. ^**P < 0.01 ^***P < 0.001 vs. Control

Fig. 6

A ELISA was carried out to determine the levels of IL-1β, LDH and IL-18 in cells. B Western blot was performed to determine the expression of pyroptosis-related proteins (GSDMD, NLRP3 and caspase-3) in cells. ^**P < 0.01 ^***P < 0.001 vs. Control