2.Results
2.1 Knocking out sdrc can inhibit the formation of MRSA biofilm
Through genetic engineering technology, we successfully knocked out the SDRC protein sequence of Staphylococcus aureus. As shown in Figure Attachment 1, PCR amplification and agarose gel electrophoresis results show that the SDRC protein of Staphylococcus aureus was successfully knocked out. To investigate the effect of sdrc knockout on the formation of MRSA biofilm, we first validated the purchased KO sdrc. RT-PCR results showed extremely low expression of sdrc in the KO sdrc group, indicating that the purchased knockout bacteria were correct and could be used for subsequent experiments (Figure 1A). Next, we plotted the growth curves of MRSA and KO sdrc at 1h, 2h, 3h, 4h, 5h, 7h, 9h, 12h, 18h, and 24h. The results showed that knocking out sdrc had no effect on the growth level of MRSA (Figure 1B), but there were invasive virulent pathogenic factors on the biofilm of MRSA α- The expression levels of HL and PSM did significantly decrease (Figure 1C). The results of crystal violet staining showed that the knockout of sdrc could weaken the formation of MRSA biofilm (Figure 1D), and the same results were obtained by scanning electron microscopy. The surface of the MRSA group was rough, and the bacterial membrane migrated towards the blank, showing a trend towards biofilm formation, while the KO sdrc group had a smooth surface and reduced bacterial count (Figure 1E). It indicates that sdrc knockout can inhibit the formation of MRSA biofilm.
2.2 Knocking out sdrc can alleviate the inhibition of MRSA on the differentiation of BMSCs into osteoblasts
Next, we examined the effect of sdrc knockout on the inhibition of BMSCs differentiation into osteoblasts by MRSA. Firstly, we examined the median lethal rate of MRSA on BMSCs cells, and the results showed an IC50 of 161.5 moi (Figure 2A). Next, we intervened with 161.5 mol of MRSA and KO sdrc on BMSCs cells to investigate the effect of sdrc knockout on the inhibition of BMSCs differentiation into osteoblasts by MRSA. The osteoblast induced differentiation group was used as a positive control, and cell culture was shown in Figure 2B. After 24 hours of intervention, CCK8 results showed that both MRSA and KO sdrc restricted the proliferation ability of BMSCs, but MRSA was more significant than KO sdrc (Figure 2C), and ALP staining results showed that, Both MRSA and KO sdrc can reduce the positive rate of ALP (there may be errors in the results here, which need to be verified multiple times) (Figure 2D and 2E). The RT-PCR results are similar to CCK8, and MRSA significantly inhibits the specific markers of osteogenic differentiation Runx2, OSX, ALP, and osteocalcin compared to KO sdrc (Figure 2F). Finally, we detected the expression of Runx2, OSX, ALP, and osteocalcin using IF experiments and obtained the same results (Figure 3A-E). It indicates that knocking out sdrc can alleviate the inhibition of MRSA on the differentiation of BMSCs into osteoblasts.
2.3 Knocking out sdrc can alleviate the progression of osteomyelitis
To investigate the impact of sdrc knockout on the progression of osteomyelitis in vivo, we first constructed an osteomyelitis model infected with MRSA. As shown in Figure 4A-B, 1 × 108and 1 × The difference in osteomyelitis caused by 109CFU/mL MRSA is not significant, so we will choose 1 in the future × 108 CFU/mL MRSA modeling. Research has shown that compared to KO sdrc alone intervention, MRSA+KO sdrc mixed intervention can more effectively alleviate the progression of osteomyelitis. Therefore, we also set up an MRSA+KO sdrc mixed intervention group, where serum C-reactive protein is an inflammatory marker protein. The CRP content test results showed that compared with KO sdrc alone intervention, the MRSA+KO sdrc mixed intervention did not reduce the release of CRP (Figure 4D).
Next, we examined the impact of knocking out sdrc on the progression of osteomyelitis. HE results showed that the control group had normal morphology and intact cortical structure without pathological changes; In the MRSA group, a large number of inflammatory cells infiltrated the bone marrow cavity, and the bone cortex at the drilling site failed to heal; A small amount of inflammatory cells and new bone formation were observed in the KO sdrc group. In the MRSA+KO sdrc group, there was bone destruction and a large amount of inflammatory cell infiltration (Figure 5A). Culture of wound exudates in each group revealed that compared to the Control group, the MRSA group had the highest bacterial density, followed by the KO sdrc group, and the MRSA+KO sdrc group had the lowest density (Figure 5B). The ELISA results showed that the KO sdrc group significantly inhibited the inflammatory factors IL-6 and IL-1 β And TNF- α The MRSA+KO sdrc group did not achieve the expected results (Figure 5C). Indicating that knocking out sdrc can alleviate the progression of osteomyelitis.
2.4 Knocking out sdrc can alleviate the inhibition of bone formation by MRSA
To investigate the effect of knocking out sdrc on the inhibition of bone formation by MRSA, we detected the expression of bone formation marker proteins Runx2, OSX, and ALP using IHC. The results showed that compared with the MRSA group, the expression of Runx2, OSX, and ALP in the KO-sdrc group was significantly increased; Compared with the KO sdrc group, the expression of Runx2, OSX, and ALP in the MRSA+KO sdrc group decreased (Figure 6A-C). It indicates that knocking out sdrc can alleviate the inhibition of bone formation by MRSA, while the mixed intervention of MRSA+KO sdrc cannot more effectively alleviate the inhibition of bone formation by MRSA.
2.5 Transcriptome sequencing reveals that knocking out sdrc alleviates osteomyelitis progression by promoting Myh7 expression
We performed transcriptome sequencing on three right tibia tissue samples from each of WT, MRSA, KO sdrc, and MRSA+KO sdrc mice, with a threshold setting of | log2 (FoldChange | ≥ 1, and pvalue<0.05). The results showed that compared with the WT group (Figure 7A (a)), a total of 457 significantly differentially expressed genes were screened, including 138 upregulated genes and 319 downregulated genes. Compared with the KO sdrc group and the WT group (Figure 7A (b)), a total of 290 significantly differentially expressed genes were screened, including 180 upregulated genes and 110 downregulated genes. Compared with the KO sdrc group and MRSA group (Figure 7A (c)), a total of 513 significantly differentially expressed genes were screened, including 312 upregulated genes and 201 downregulated genes. Comparing the KO sdrc+MRSA group with WT (Figure 7A (d)), a total of 628 significantly differentially expressed genes were screened, including 202 upregulated genes and 426 downregulated genes. We will use the Top100 gene with the lowest q value as an example of a heatmap. Venn analysis was performed on the significantly differentially expressed genes in the MRSA group Vs WT group, KO sdrc group Vs WT group, and KO sdrc group Vs MRSA group, as shown in Figure 7B. A total of 5 common genes were screened to identify the reasons for downregulation in the WT group Vs MRSA group, upregulation in the WT group Vs KO sdrc, and upregulation in the KO sdrc group Vs MRSA group. It was found that these five genes Myh7, Tnnc1, Myl2, Igkv4-59, and CT010467 all met the conditions (Figure 7B). Through literature review, it can be concluded that Myh7 and Tnnc1 genes are associated with skeletal muscle. After removing the new genes Igkv4-59 and CT010467, this study focuses on three genes: Myh7, Myl2, and Tnnc1, and their expression multiples are shown in 7C.
Next, we validated the sequencing results. RT-PCR results showed that all three genes, Myh7, Myl2, and Tnnc1, did not match the sequencing results (compared to the WT group, they were all down regulated in the MRSA group and up regulated in the KO sdrc group) (Figure 8A). The WB results showed that only Myh7 followed the trend (Figure 8B), and we also validated it with IHC, which was the same as the WB results. Therefore, we speculate that the knockout of sdrc may be by increasing the expression of Myh7, Relieve the progression of osteomyelitis. Compared with the KO sdrc group, the MRSA+KO sdrc mixed intervention group did not increase the expression of Myh7.
2.6 In vitro validation: Knocking out sdrc can promote bone formation by promoting Myh7 expression
To further verify that knocking out sdrc promotes bone formation by promoting Myh7 expression, we constructed a stable osteoclast transfection strain with MYH7 interference (Figure Attachment 2). The results showed that compared with the KO sdrc+sh NC group, the KO sdrc+sh Myh7 group had a decrease in osteoclast proliferation ability (Figure 9A), and the expressions of Myh7, Runx2, OSX, ALP, and osteocalcin were significantly reduced (Figure 9B). The positive rates of ALP and alizarin red staining were also significantly reduced (Figure 9C-D). It indicates that knocking out sdrc indeed promotes bone formation and alleviates the progression of osteomyelitis by promoting MYH7 expression.