Staphylococcus aureus virulence factors are predominantly categorized into adhesins, toxins, and immune evasion factors (Becker, 2018). The production of these virulence factors varies depending upon the environment. In diabetic wounds, hyperglycemia occurs and promotes bacterial colonization. A comparative analysis of the expression profiles of three types of virulence factors—adhesins, toxins, and immune evasion factors—in wounds from in mice exhibiting hyperglycemic and normoglycemic conditions was conducted in vitro by using medium with and without in a high-sugar, respectively.The results revealed a substantial increase in the expression levels of adhesin genes, including aaa, clfB, fnbA, isdA, isdC, mntC, sdrH, tarK, eap, and emp, in S. aureus subjected to an elevated glucose concentration (Figure 1A). Concurrently, the expression of toxin genes such as hlgB, ssl5, adsA, aur, lgt, and nuc, and the immune evasion factor essb also markedly upregulated (Figures 1B,C). With a mouse DFU model, S. aureus markedly upregulated the expression of some adhesin genes, including isdB, sdrH, icaA, icaC, and icaD (Figure 2A). A significant increase in the expression of toxin genes like ssl5, hlb, and nuc, and the immune evasion factor essb was also observed (Figures 2B,C). These findings demonstrated that a high-glucose environment induces the expression of various virulence factor genes such as sdrH and icaA-C in S. aureus. These upregulated genes may be subjected to manipulation of host immune response.

To further investigate the roles of sdrH and icaA-C in S. aureus-caused DFUs, the related gene mutants were generated with homologous recombination strategy. Successfully, the sdrH and icaA-C single mutant as well as the sdrH/icaA-C double mutant strain were constructed (Figures 3A,B, 4A,B).

Aureobasidium spp. were cultured into high and low sugar DMEM medium, and biofilm formation was measured after 48 h of incubation. By scanning electron microscopy, we found that the presence of the sdrH gene in the high sugar state could help S. aureus to form more biofilms during the proliferation process (Figure 5). While comparing the biofilm expression of the four S. aureus different strains in their low sugar condition, we found that the presence of the sdrH gene could help S. aureus form more biofilms, but there was a gap compared to the amount of sdrH gene promoting the biofilm expression of S. aureus in the high sugar culture condition. In contrast, the icaA-C gene mutation had no significant effect on biofilm formation in S. aureus regardless of the environment. Therefore, we conclude that S. aureus can form biofilms through the expression of the sdrH gene, and that glucose in a high sugar environment promotes the up-regulation of the expression of the sdrH gene, assisting in the production of more biofilms and enhancing its colonisation ability.

The sdrH gene encodes the serine-aspartic repeat protein H (SdrH), which belongs to a family of staphylococcal adhesion proteins containing a serine-aspartic repeat (SDR) domain and is essential for Staphylococcus aureus infection (Kim et al., 2021). The function of S. aureus adhesins in DFUs remains elusive. Experimental outcomes indicated that the NewmanΔsdrH mutant exhibited substantial attenuation of pathogenicity relative to the wild-type strain, as evidenced by the accelerated healing of back wounds in diabetic mice (Figures 6A,B). The result also demonstrated the markedly enhanced rate of wound closure in these mice (Figure 6C). The SdrH significantly augmented the colonization of S. aureus in the wounds (Figure 7). Overall, throughout the infection process caused by Staphylococcus aureus, the sdrH gene enhances the bacteria’s adhesion capabilities, resulting in a significant increase in bacterial colonization within the infected wound. This escalation in colonization subsequently affects the wound’s healing process.

Expression of the icaA-C gene cluster leads to the production of a polysaccharide intercellular adhesin (PIA)-dependent biofilm (Cramton et al., 1999), which enhances the colonization capabilities of S. aureus. Our results showed that there was no significant difference in wound size between mice infected with the NewmanΔicaA-C and the wild-type strain. To ascertain whether the concurrent elimination of icaA-C and sdrH would reduce the infective capacity of Staphylococcus aureus, a double mutant NewmanΔicaA-C/ΔsdrH was generated. Remarkably, a reduction in the wound healing duration in mice infected with NewmanΔicaA-C/ΔsdrH was observed (Figure 6). Furthermore, the bacterial burden in the wounds was substantially reduced compared to that observed in the Newman- and NewmanΔsdrH-infected cohorts, as shown in Figure 7. The findings showed that the simultaneous knockout of the sdrH and icaA-C genes can mitigate the colonization of S. aureus within wounds, diminish wound infections, enhance the host’s repair capacity, and facilitate wound healing.

The histopathological examination of mouse back wounds on day 5 post-infection was performed. Masson’s trichrome staining results revealed that the protein sdrH mitigated the production of type I and type III collagen fibers, abrogated the differentiation of fibroblasts, and perturbed the healing and regenerative processes of host wounds. Moreover, immunofluorescence assay results indicated that SdrH influences angiogenesis during wound mending and re-epithelialization, which in turn delays wound closure by impeding the recovery of vascular networks. Additionally, greater COL1 and COL3 collagen fiber production was observed, and enhanced neovascularization was evident around the wound site (Figure 8). These findings indicate that icaA-C may aid sdrH in modulating the host’s immune response during Staphylococcus aureus infection of wounds, which in turn promotes the pathogen’s ability to colonize the wound and slows wound healing. Collectively, the deletion of the sdrh gene may affect the expression of specific pathways, reduce the colonization ability and virulence of Staphylococcus aureus in wounds, and enhance the immune system’s capacity to eliminate the bacteria. This alteration can subsequently modify the intracellular environment of the host wound, facilitating the regeneration of wound collagen and microvasculature, thereby accelerating the healing of the host wound surface.

In the present study, the primary objective was to isolate the Newman strain of Staphylococcus aureus (NCTC 8178, ST1/agr I) from throat swab samples obtained from patients experiencing secondary infections associated with tuberculous osteomyelitis (Duthie and Lorenz, 1952; Missiakas and Schneewind, 2013). The culture was subsequently inoculated and incubated at 37°C following the reanimation of Staphylococcus aureus, which had been preserved at −80°C, on Trypticase Soy Broth (TSB) agar until distinct bacterial colonies formed. Thereafter, the selected bacterial colonies were cultured overnight under conditions of agitation at 200 rpm.

Female BALB/c mice, aged 6 weeks, were procured from the Experimental Animal Center of Army Medical University (AMU). The study was conducted in adherence with the guidelines outlined in the Regulations on the Administration of Experimental Animals, as ratified by the State Council of the People’s Republic of China. Following 1 week of adaptive feeding, the mice were progressively transitioned to a diet high in fat and sugar content over 30 days, during which their weight fluctuations were meticulously monitored. Streptozotocin (STZ) was subsequently administered at a dosage of 60 mg per kilogram of body weight over a span of five consecutive days to establish a diabetic mouse model. Prior to each injection, the mice were fasted for a 12-h period. Plasma glucose levels were assessed on the 7th day post-injection, and a blood glucose level ≥16.7 mmol/L was deemed indicative of the establishment of a diabetic mouse model (Vågesjö et al., 2018). The dorsal regions of each mouse (both the control and diabetic mice) were subsequently shaved, and a full-thickness skin lesion of approximately 10 mm in diameter was created. This wound was subsequently sutured with a rubber ring to prevent inherent skin contraction (Michaels et al., 2007). A suspension of Staphylococcus aureus Newman, containing 1 × 10⁷ colony-forming units (CFUs), was instilled into the skin wounds of both diabetic and control mice.

On the fifth day post-infection, both diabetic and control mice were euthanized. The wound tissue and pus collected from the mice were processed using the TRIPURE method. Following homogenization and grinding of the samples, bacterial RNA was extracted using chloroform and isopropyl alcohol. The precipitate was analyzed to confirm that the OD260/280 and OD260/230 ratios exceeded 1.8. After eliminating genomic contamination from the validated RNA, cDNA reverse transcription was performed using the Invitrogen SuperScript IV cDNA synthesis kit. To replicate the host body’s growth conditions for S. aureus, the bacteria were concurrently introduced into standard DMEM and high glucose DMEM (4.5 g/L). RNA was then extracted from bacterial cultures during the logarithmic growth phase using an RNA extraction kit. The obtained RNA was utilized with the RevertAid First Strand cDNA Synthesis Kit from ThermoFisher. Specifically, 5 μg of RNA was taken and mixed with 1 μL of Random Hexamer primer, followed by the addition of ribozyme-free water to a final volume of 12 μL. After incubating at 65°C for 5 min, 4 μL of 5X Reaction Buffer was added, along with 1 μL of RiboLock RNase Inhibitor (20 U/μL), 2 μL of 10 mM dNTP Mix, and 1 μL of RevertAid M-MuLV RT (200 U/μL), bringing the total volume to 20 μL. The mixture was thoroughly mixed and incubated at 25°C for 5 min, followed by an incubation at 42°C for 60 min. After this period, the transcribed cDNA can be obtained. The concentration of the resulting cDNA was quantified with a spectrophotometer, diluted with DEPC water to achieve a uniform concentration, and utilized as a template to analyze the expression levels of forty genes associated with adhesins, toxins, and immune evasion. Primers can be found in the Supplementary materials. Primers can be found in the Supplementary materials. To create a Staphylococcus aureus Newman strain with gene knockouts of the upregulated genes, the temperature-sensitive E. coli-Staphylococcus aureus shuttle plasmid pBT2 was employed based on primer design surrounding the target gene (Brückner, 1997). Each resulting mutant underwent thorough verification through PCR amplification and subsequent sequencing. The sdrH gene knockout strain was designated as NewmanΔsdrH. In conclusion, this study successfully constructed the icaA-C gene cluster knockout strain NewmanΔicaA-C and the icaA-C/sdrH double knockout strain NewmanΔicaA-C/ΔsdrH.

RT-qPCR detection was conducted using the BioRad Sso Advanced Universal SYBR® Green master mix. The obtained cDNA was diluted 100-fold with DEPC-treated water and utilized as a template. The SYBR® Green qPCR mixture was prepared, and three biological replicates were performed, with each sample set up in triplicate wells. For each reaction, 10 μL of 2 × Sso Advanced SYBR® Green master mix, 1 μL of each upstream and downstream primer, 1 μL of the diluted template, and 7 μ L of DEPC-treated water were combined. Use the CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad of the United States, 1,855,201). The thermal cycling conditions included pre-denaturation at 95 ° C for 3 min, denaturation at 95 ° C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 30 s, with a total of 42 cycles. The dissociation curve was generated by increasing the temperature from 58°C to 95°C in increments of 0.5°C, maintaining each increment for 5 s to measure changes in fluorescence intensity. A single peak in the dissociation curve at the annealing temperature indicates high specificity.

Four different strains of S. aureus were inoculated into Brain Heart Infusion (BHI) slant medium respectively, and cultured for 18–24 h, then washed down the bacterial foetus on the slant to form bacterial suspension with PBS (phosphate buffer solution), washed with PBS for 3 times, and then the 96-well plate was 100ul per well, with 3 replicate wells for each bacterium, and its OD value was measured in the enzyme marker at 570 nm, and then the bacterial suspension was configured as 0.5OD with DMEM The medium (high sugar and low sugar) was configured as 0.5OD bacterial suspension. 24-well plates were placed in sterile slides and 0.5 mL of 0.5OD bacterial suspension was added to each well, and the biofilm was prepared by incubation for 6 days at 37°C. The medium in the wells was washed out after 6 days, and washed with PBS for 2–3 times, and then 2.5% glutaraldehyde was added to each well for pre-fixation, and then 1% osmium tetroxide for post-fixation was added to the wells. After washing the fixative in PBS solution at pH 7.4, it was fully dehydrated by graded ethanol (30, 50, 70, 90, 100% ethanol twice) and finally critical point dried and coated for observation. Using Hitachi SU-8010 field emission scanning electron microscope to observe the biofilm and take screenshots for subsequent statistics.

Following the establishment of the mouse model of DU, the experimental cohort of diabetic mice, consisting of 10 individuals, was inoculated with a gene knockout strain at a density of 1 × 10⁷ CFU. Concurrently, 10 control mice were infected with the Newman strain at the same dose, and subsequent colonization by the wild-type strains was permitted. Continuous monitoring of wound closure was conducted, with images being captured at 3, 5, 7, 9, 11, and 14 days post-bacterial colonization to determine the wound healing velocity. Tissue samples from the wounds were collected on days 1, 3, 7, and 14. They were subsequently fixed in a 4% tissue fixative solution for 48 h, followed by paraffin embedding and staining with hematoxylin–eosin (HE) and Masson’s trichrome. Immunofluorescence was used to detect the expression of CD31 (with an anti-CD31 mouse monoclonal antibody), while immunohistochemistry was used to detect collagen expression (with a mouse anti-collagen I antibody and an anti-collagen type III polyclonal antibody). Immunofluorescence was employed to assess vascular formation through ImageJ analysis. The image to be analyzed is opened in ImageJ, converted to 8-bit format, and bright field images are processed to identify branch points and intersection points for statistical analysis. A similar approach is utilized for immunohistochemistry. The results of immunohistochemistry are analyzed using ImageJ to quantify collagen area and vascular formation. In this process, the image is opened in ImageJ, and image deconvolution is applied to separate the DAB and hematoxylin channels. The DAB color development signal window is selected, and the grayscale signal is converted to optical density values. The upper and lower limits of the threshold are manually adjusted to encompass the ideal area, allowing for quantitative data analysis.

All experiments were conducted a minimum of three times in triplicate, with each sample representing a collective from at least three distinct culture dishes or three individual samples. Statistical analyses were performed via GraphPad Prism version 9.3 (GraphPad Software, Inc., USA). An unpaired t test was used to compare the mean values between the two groups. Furthermore, two-way ANOVA was utilized to assess the differences in cytokine levels and wound healing rates, whereas one-way ANOVA was adopted for the comparison of skin wound bacterial loads. The results are presented as the means ± standard deviations (SDs), with a significance level of p < 0.05.