Abstract
The Staphylococcus aureus (S. aureus) SaeRS two-component system (TCS) regulates over 20 virulence factors. While its impact on chronic infection has been thoroughly discussed, its role in the early stage of infection remains elusive. Since macrophages serve as the primary immune defenders at the onset of infection, this study investigates the influence of SaeRS on macrophage functions and elucidates the underlying mechanisms. Macrophage expression of inflammatory and chemotactic factors, phagocytosis, and bactericidal activity against S. aureus were assessed, along with the evaluation of cellular oxidative stress. SaeRS was found to impair macrophage function. Mechanistically, SaeRS inhibited NF-κB pathway activation via toll-like receptor 2 (TLR2). Its immune-modulating effect could partially be explained by the strengthened biofilm formation. More importantly, we found SaeRS compromised macrophage immune functions at early infection stages even prior to biofilm formation. These early immune evasion effects were dependent on bacterial clumping as cytokine secretion, phagocytosis, and bactericidal activity were repaired when clumping was inhibited. We speculate that the bacterial clumping-mediated antigen mask is responsible for SaeRS-mediated immune evasion at the early infection stage. In vivo, ΔsaeRS infection was cleared earlier, accompanied by early pro-inflammatory cytokines production, and increased tissue oxidative stress. Subsequently, macrophages transitioned to an anti-inflammatory state, thereby promoting tissue repair. In summary, our findings underscore the critical role of the SaeRS TCS in S. aureus pathogenicity, particularly during early infection, which is likely initiated by SaeRS-mediated bacterial clumping.
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Introduction
Staphylococcus aureus (S. aureus), one of the most common clinical pathogens, causes various infectious diseases, including osteomyelitis, bacteremia, and pneumonia1,2,3. During the early stage of infection, the innate immune system, represented by neutrophils and macrophages, generates immune responses to counter the invasion of S. aureus4. However, virulence factors secreted by S. aureus facilitate bacterial invasion and impair host immune functions in a variety of ways, including suppressing neutrophil recruitment, impeding macrophage bactericidal activity, and interfering with complement system activation5,6,7,8,9. The production of virulence factors by S. aureus and its adaptability to external stimuli are intricately governed by complex regulatory networks, including the SarA family, alternative sigma factor B, and two-component systems (TCS), such as AgrAC, SrrAB, and SaeRS10,11,12.
Regulating the expression of multiple virulence factors, SaeRS TCS is encoded within the sae locus, which consists of four genes, saeP, saeQ, saeR, and saeS, and two promoters (P1 and P3)13,14,15. saeS encodes a histidine kinase sensor, whereas saeR encodes a response regulator. Auxiliary proteins encoded by saeP and saeQ bind to those encoded by saeS and activate their phosphatase activity16. In response to environmental stimuli, saeS undergoes histidine residue autophosphorylation, subsequently activating a signaling cascade within the SaeRS TCS. The cascade transfers phosphate groups to aspartate residues in saeR. Afterwards, phosphorylated saeR binds to specific recognition sequences in the promoter regions of target genes, activating their transcription17.
Although the impact of SaeRS on biofilm formation and its pathogenicity contribution to chronic infections has been widely discussed18,19,20,21, studies regarding its influence on early infection and underlying mechanisms are limited. In our previously published scRNA-seq data of S. aureus implant infection, we found macrophages accumulate at the infection site during the early stage of S. aureus infection and decrease in chronic state, suggesting a significant function of macrophage in early infection22. Neutrophils and macrophages drive the primary immune response at the early stage of infection. While previous studies have extensively investigated the impact of SaeRS TCS on neutrophils, it remains largely unexplored the effects of SaeRS TCS on macrophages, and the underlying mechanisms are poorly understood as well23,24,25,26,27.
In this study, SaeRS was found to impair macrophage function. Mechanistically, SaeRS inhibited macrophage NF-κB pathway activation via toll-like receptor 2 (TLR2). Its immune-modulating effect could partially be explained by the strengthened biofilm formation. More importantly, we found SaeRS compromised macrophage immune functions at early infection stages even prior to biofilm formation, this was demonstrated to be dependent on bacterial clumping. This study underscored the crucial role of SaeRS in S. aureus pathogenicity, especially in the early stage of infection.
Results
SaeRS inhibits macrophage immune regulatory activities
To explore the impact of SaeRS on the secretion profiles of pro-inflammatory factors and chemokines, macrophages were separately co-cultured with the wild type (WT) or ΔsaeRS strain for 2 h. Additionally, fenoprofen, identified by our team as a SaeR inhibitor via structure-based virtual screening, can bind directly to the promoter binding region of the SaeR protein, inhibiting SaeRS TCS regulation of virulence genes without affecting bacterial growth28. Therefore, the WT strain pretreated with fenoprofen was used as a parallel control to the ΔsaeRS strain.
We observed a significant increase in the levels of several cytokines responsible for macrophage and neutrophil recruitment (CCL3 and CXCL2) and activation (tissue necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6) at both the RNA and protein levels (Fig. 1C‒L). Inducible nitric oxide synthase (NOS2) synthesis in macrophages is a key bactericidal mechanism, catalyzing nitric oxide (NO) production from arginine, which acts directly against bacteria and regulates immune networks29,30. Macrophages stimulated by the ΔsaeRS strain or fenoprofen-treated WT strain exhibited significantly increased transcription levels of NOS2 and a corresponding increase in NO production (Fig. 1A, B). Comparable results were also found in mouse macrophages (Supplementary Fig. 1). These results indicate that mutation in the SaeRS TCS significantly enhances macrophage immune regulatory capabilities.
A, C, E, G, I, K RT-qPCR results of NOS2 (A), IL-1β (C), IL-6 (E), TNF-α (G), CXCL2 (I), and CCL3 (K) after various treatments. B, D, F, H, J, L Concentrations of NO (B), IL-1β (D), IL-6 (F), TNF-α (H), CXCL2 (J), and CCL3 (L) after various treatments. n = 3 per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NOS2, nitric oxide synthase; IL, interleukin, TNF-α, tissue necrosis factor-α.
SaeRS compromises the phagocytic and bactericidal abilities of macrophages
Phagocytosis of pathogens and dead cells is a crucial function of macrophages in host defense and tissue homeostasis. We further assessed the impact of the SaeRS on the phagocytic and bactericidal abilities of macrophages. Immunofluorescence and flow cytometry analyses revealed an increased engulfment of green fluorescent protein (GFP)-labeled ΔsaeRS and fenoprofen-treated WT strains by macrophages compared to the WT strain (Fig. 2A–C and Supplementary Fig. 2A–C). Additionally, bacterial viability within macrophages significantly declined after 2, 12, and 24 h of phagocytosis for both ΔsaeRS and fenoprofen-treated WT strains, as assessed using the spread plate method (SPM), indicating that SaeRS impairs macrophage bactericidal capacity (Fig. 2D–F and Supplementary Fig. 2D–G). These findings collectively suggest that the SaeRS TCS detrimentally affects the phagocytic and bactericidal capabilities of macrophages.
A Representative images of macrophages ingesting different groups of S. aureus (Scale bar, 10 μm). B, C Evaluation of the phagocytosis ability of macrophages via flow cytometry (n = 3 per group). D–F Assessment of bactericidal capacity of macrophages after 2, 12, and 24 h of phagocytosis (n = 6 per group). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD.
SaeRS hinders macrophage invasion and clearance of biofilms
S. aureus forms dense biofilms as a defensive barrier, secreting extracellular polymeric substances to shield itself from antibiotics and immune system attacks, which poses a challenge for macrophages to penetrate and eliminate bacteria, leading to persistent infections31,32,33. Confocal laser scanning microscopy (CLSM) analysis revealed significant differences in biofilm characteristics, with biofilms from ΔsaeRS and fenoprofen-treated WT strains showing increased porosity and reduced thickness compared to those from the WT strain (Fig. 3A and Supplementary Fig. 3). Crystal violet experiments further demonstrated a noteworthy reduction in biomass of ΔsaeRS biofilms (Fig. 3B), corroborated by SPM results (Fig. 3D). In co-cultures with macrophages, biofilms with saeRS mutation or inhibition exhibited enhanced susceptibility. The intact and dense structure of the WT biofilm resulted in fewer macrophages adhering to and invading the biofilm compared to those formed by ΔsaeRS and fenoprofen-treated WT strains (Fig. 3C and Supplementary Fig. 4A). Moreover, macrophages demonstrated increased bactericidal capabilities against bacteria within the ΔsaeRS biofilms, leading to substantially lower bacterial survival rates for ΔsaeRS and fenoprofen-treated WT strains (Fig. 3D and Supplementary Fig. 4B, C). Taken together, the SaeRS TCS contributes to the dense structure of biofilms, impairing macrophage adhesion, invasion, and bactericidal activities against biofilms.
A Representative CLSM images of biofilms formed by different strains of S. aureus (Scale bar, 200 μm and 20 μm). B Quantification of the biomass within the biofilm via the crystal violet assay (n = 6 per group). C Representative CLSM images of macrophages co-cultured with biofilms formed by different strains of S. aureus (scale bar, 200 μm). D Bacterial loads and survival rates within different biofilm groups before and after co-culturing with macrophages (n = 6 per group). ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD. CLSM, confocal laser scanning microscopy.
SaeRS suppresses macrophage oxidative stress levels
The bactericidal activities of macrophages rely on intracellular ROS34,35. Here, the C11 BODIPY 581/591 probe, DCFH-DA probe, and GSH tracer were employed to assess levels of lipid peroxidation, ROS, and glutathione (GSH), respectively, as conventional markers of oxidative stress36,37.
As depicted in Fig. 4A and Supplementary Fig. 5A, both mouse Raw264.7 and human THP-1 macrophages infected with the WT strain exhibited increased lipid peroxidation, as evidenced by a reduced intensity of red fluorescence in the reduced state and an increased intensity of green fluorescence in the oxidized state. Correspondingly, the level of the lipid peroxidation product, malondialdehyde (MDA), was elevated (Fig. 4B and Supplementary Fig. 5B). In contrast, lipid peroxidation levels in macrophages were further heightened after co-culturing with ΔsaeRS or fenoprofen-treated WT strain, accompanied by an increase in MDA production. Similarly, macrophages infected with ΔsaeRS or fenoprofen-treated WT strain displayed significantly higher levels of ROS compared to those infected with the WT strain (Fig. 4C, D and Supplementary Fig. 5C, D). Meanwhile, cells treated with ΔsaeRS or fenoprofen-treated WT strain exhibited lower GSH levels and a reduced GSH/GSSG ratio (Fig. 4E‒G and Supplementary Fig. 5E-G). These results confirm that SaeRS reduces macrophage oxidative stress levels, which elucidates the observed increase in the production of inflammatory factors and enhanced bactericidal activities in macrophages infected with ΔsaeRS or fenoprofen-treated WT strain.
A Representative fluorescence staining images depicting lipid peroxidation levels in macrophages after co-culturing with different strains of S. aureus (Scale bar, 20 μm). Oxidation of the probe results in a fluorescence shift from red to green. B Relative MDA content within macrophages of each group. C Fluorescence intensity of macrophages from different groups after DCFH-DA staining. D Representative fluorescence staining images of intracellular ROS in macrophages (Scale bar, 20 μm). The DCFH-DA probe emits green fluorescence when combined with ROS. E Representative fluorescence staining images of GSH stained with a GSH tracer in macrophages (Scale bar, 40 μm). Probes bound to GSH emit red fluorescence, and unbound probes emit green fluorescence. F Total glutathione content in macrophages from different groups. G GSH/GSSG ratio in macrophages. n = 3 per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD. MDA, malondialdehyde; ROS, reactive oxygen species; DCFH-DA, dichlorodihydrofluorescein diacetate; GSH, glutathione; GSSG, glutathione disulfide.
saeRS mutation enhances the activation of the NF-κB pathway in macrophages
To further explore the mechanisms responsible for the observed alterations in macrophage immune phenotype, transcriptome sequencing of macrophages infected with WT or ΔsaeRS strain was performed. Compared with the WT strain, ΔsaeRS strain infection upregulated numerous inflammatory factors and chemokines, consistent with our earlier findings (Fig. 5A, B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further indicated that multiple inflammation-related signaling pathways were largely upregulated following ΔsaeRS strain infection. Among these, the NF-κB signaling pathway exhibited the most significant changes, suggesting that this pathway may be the primary driver of the enhanced immune response observed in macrophages (Fig. 5C). Moreover, gene ontology (GO) enrichment analysis identified several infection-immunity-related pathways, supporting the KEGG enrichment results (Fig. 5D). This was corroborated by gene set enrichment analysis (GSEA) (Fig. 5E). Western blotting and immunofluorescence staining for the key factor, p65, in the NF-κB pathway, provided additional validation (Fig. 5F‒H). Mouse macrophages also exhibited the same trend (Supplementary Fig. 6). Following WT strain infection, macrophages displayed increased p65 phosphorylation and enhanced nuclear translocation. In contrast, ΔsaeRS strain infection resulted in even higher levels of p65 phosphorylation and increased nuclear translocation. In summary, these findings indicate that, compared with the WT strain, ΔsaeRS infection further amplifies the activation of the NF-κB pathway in macrophages, resulting in a robust immune response to infection.
A Volcano plots of differentially expressed mRNAs in macrophages co-incubated with mutant or WT strain. B Heat map constructed by the top 25 upregulated and downregulated mRNAs. Warm and cold colors indicate upregulated and downregulated mRNAs, respectively. C, D Top 20 enriched KEGG pathways and GO terms of differentially expressed mRNAs between macrophages co-incubated with mutant or WT strain. E GSEA of NF-kB pathway in macrophages. F, G Western blotting analysis of p65 phosphorylation levels (n = 3 per group). H Representative immunofluorescence staining images depicting the nuclear translocation of NF-κB p65 in various macrophage groups (Scale bar, 5 μm). * p ≤ 0.05, ** p ≤ 0.01. Data are presented as means ± SD. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis.
SaeRS promotes bacterial clumping, impairing TLR2 recognition, NF-κB pathway activation, and immune response in macrophages
TLR2 is the primary pattern recognition receptor (PRR) responsible for S. aureus infections38. Studies have shown that TLR2 plays a crucial role in recognizing S. aureus and activating immune responses39,40,41. We hypothesized that TLR2 is the initiation point for NF-κB pathway activation in macrophages induced by ΔsaeRS strain. To test this hypothesis, we pre-incubated macrophages with neutralizing antibodies against TLR2 and subsequently co-cultured them with various bacterial strains to assess relevant signaling pathways and immune phenotypes. Upon TLR2 receptor neutralization, a significant reduction in phosphorylation levels of p65 was observed in macrophages infected with the ΔsaeRS strain or fenoprofen-treated WT strain (Fig. 6A and B). Additionally, the nuclear translocation of phosphorylated p65 was relatively diminished (Fig. 6C).
A, B Western blot analysis of p65 phosphorylation levels. C Representative images of p65 nuclear translocation in different treatment groups (Scale bar, 5 μm). D Gene expression and product concentrations of inflammatory and chemotactic factors in THP-1 cells after different treatments. E Representative images of THP-1 macrophages engulfing S. aureus (Scale bar, 10 μm). F The phagocytosis rates of THP-1 cells. n = 3 per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD.
Furthermore, the transcription and secretion of inflammatory factors and chemokines by macrophages, pretreated with neutralizing antibodies and co-cultured with ΔsaeRS or fenoprofen-treated WT strain, were notably reduced (Fig. 6D). This indicates the suppression of macrophage immunoregulatory functions. We employed fluorescence staining and flow cytometry to evaluate macrophage phagocytosis, while bactericidal activity and bacterial proliferation within macrophages were quantified using SPM. The results demonstrated significantly inhibited phagocytosis and compromised bactericidal activity by TLR2-neutralized macrophages against ΔsaeRS or fenoprofen-treated WT strain, compared with macrophages without antibody treatment (Fig. 6E, F). Notably, similar results were observed in mouse macrophages, suggesting that the impact of ΔsaeRS and fenoprofen-treated WT strains on the NF-κB pathway in macrophages and the underlying mechanisms are consistent across different species (Supplementary Fig. 7-9).
Macrophages can recognize antigens of S. aureus through TLR2, initiating the immune response42,43. However, S. aureus employs various strategies to evade immune recognition by macrophages, with one typical approach being the formation of clumps through binding extracellular proteins to prevent antigen exposure44. Using fetal bovine serum (FBS) to simulate extracellular proteins in the infection microenvironment, we observed that WT and complemented strains form bacterial clumps in the presence of FBS while remaining dispersed in environments lacking FBS. Conversely, the ΔsaeRS strain consistently remains dispersed (Fig. 7A). Consequently, macrophages stimulated by dispersed S. aureus produce higher levels of immunoregulatory factors compared to those stimulated by clustered bacteria, indicating that the enhanced exposure of antigens due to dispersion may potentially enhance the immune response of macrophages (Fig. 7B and Supplementary Fig. 10).
A Representative morphology of GFP-labeled S. aureus strains cultured in diverse environments. B S. aureus strains cultured in different environments were collected and then co-cultured with macrophages. The mRNA expression level and concentrations of CXCL2, CCL3, IL-6, and TNF-α were detected. Macrophages without any treatment were used as the control (n = 3 per group). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD.
In summary, our findings indicate that SaeRS promotes bacterial clumping, impairing the recognition of TLR2, which further hinders the NF-κB pathway, leading to changes in macrophage immune function.
saeRS mutation or inhibition promotes implant-related S. aureus infection clearance
Previous in vitro experiments have shown that SaeRS inhibits macrophage immune functions through SaeRS-mediated bacterial clumping and dense biofilm formation. Considering the coexistence of planktonic and biofilm states during S. aureus infections, especially in implant-related infections where both forms are prevalent at the interface between the implant and surrounding tissue, we investigated the impact of SaeRS on immune responses using an implant-associated infection model (Fig. 8A). Following various treatments, we observed and photographed changes in the wounds on the backs of the mice and the progression of implant infections. As depicted in Fig. 8B, a gradual increase in the size of the infection site was noted on the backs of mice infected with the WT strain, accompanied by heightened subcutaneous purulent exudate, skin ulceration, implant exposure, and implant detachment. In contrast, mice infected with the ΔsaeRS strain or treated with fenoprofen did not exhibit significant purulent exudate or skin ulceration throughout the observation period, and their implants remained relatively intact beneath the skin (Supplementary Fig. 11). Moreover, among the four groups, uninfected mice demonstrated the most rapid weight gain, followed by ΔsaeRS strain-infected and fenoprofen-treated mice. Mice infected with the WT strain exhibited the slowest and most inconsistent weight gain, indicating that ΔsaeRS strain-infected and fenoprofen-treated mice had better control of the infection within their bodies (Supplementary Fig. 12).
A Schematic illustration of implant-associated infection model and experimental procedures. B Representative images of the infection site on days 0, 4, 7, 10, and 14 after surgery in each treatment group. C, D Bacterial load of the implants and surrounding soft tissues in various groups on days 4, 7, and 14 after surgery (n = 6 per group). E Representative H & E images for each group on days 4, 7, and 14 after surgery (Scale bar, 100 μm, and 40 μm). F Representative Giemsa staining images for each group on days 4, 7, and 14 after surgery. Bacterial residue within the soft tissues is marked with red arrows (Scale bar, 20 μm, and 10 μm). ns: no significant, ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD. H & E, Hematoxylin and Eosin.
Tissue samples from the implant site were collected at 4-, 7-, and 14-day post-infection. Histological changes in the soft tissue microenvironment around the implants and residual bacterial loads were evaluated using Hematoxylin and Eosin (H&E) and Giemsa staining (Fig. 8E, F). WT strain-infected mice displayed severe tissue necrosis, inflammatory exudates, and inflammatory cell infiltration in soft tissue, particularly on days 7 and 14. In contrast, ΔsaeRS strain-infected and fenoprofen-treated mice exhibited a more pronounced inflammatory response on day 4, followed by a gradual reduction in inflammation, with a significant decrease by day 14. Furthermore, Giemsa staining revealed that although no differences in bacterial residual levels were observed among the infected mice on day 4, as the infection progressed over time, ΔsaeRS strain-infected and fenoprofen-treated mice demonstrated significantly reduced residual bacteria compared with the WT strain-infected mice on days 7 and 14. This finding was confirmed by quantitative analysis of residual bacteria in the surrounding soft tissues using SPM (Fig. 8D). S. aureus-specific immunofluorescence staining was performed to provide in vivo evidence of the clumping phenotype. By day 4 post-infection, while bacterial loads were comparable across groups, S. aureus exhibited distinct clumping in WT-infected tissues, whereas in ΔsaeRS strain-infected and fenoprofen-treated mice, the bacteria showed a more dispersed distribution.
To examine the morphological structure and quantity of bacteria adhering to the surface of the implants at different time points, scanning electron microscopy (SEM) was employed (Supplementary Fig. 14). On day 4, bacterial attachment was observed on the surfaces of all implants. By day 7, densely packed bacteria were observed on the surface of the implants in the WT infection group. Although bacterial counts on the surface of the implants in the WT infection group were slightly reduced by day 14, dense bacterial clusters were still observed. In contrast, residual bacterial counts on the implant surfaces in the ΔsaeRS strain-infected and fenoprofen-treated mice were significantly reduced at days 7 and 14, with bacteria appearing more dispersed. Quantitative analysis of the bacteria adhering to the implant surface showed a similar trend (Fig. 8C). These data suggest that ΔsaeRS strain causes less tissue damage and is more easily cleared by the host at the early stage of infection.
saeRS mutation or inhibition affects in vivo antimicrobial immune processes
The tissues surrounding the implants were collected at 4- and 14-day post-infection, allowing the evaluation of immune cell distribution and functional changes in the peri-implant immune microenvironment. Immunohistochemistry and immunofluorescence staining were employed to analyze these aspects at different time points in various treatment groups. On day 4 of infection, we observed a higher accumulation of macrophages and neutrophils in the tissue around the implants in ΔsaeRS strain-infected and fenoprofen-treated groups compared to the WT-infected group. Conversely, on day 14 post-infection, increased macrophage and neutrophil infiltration were noted around the implants in the WT-infected group, consistent with earlier H & E staining results (Fig. 9A, B).
A Representative immunohistochemical images of F4/80 within the peri-implant infected soft tissues for each group on days 4 and 14 after surgery (Scale bar, 50 μm). B Representative immunohistochemical images of Ly6G in the tissues surrounding the implants on days 4 and 14 after surgery (scale bar, 50 μm). C Representative immunofluorescence staining images of CD11b and MPO in the tissues surrounding the implants for each group on days 4 and 14 after surgery (Scale bar, 50 μm). D Representative immunofluorescence staining images of iNOS and CD206 in the tissues surrounding the implants on days 4 and 14 after surgery (Scale bar, 50 μm). E Representative ROS fluorescence staining images and the corresponding fluorescence intensity of the peri-implant soft tissues on days 4 and 14 after surgery (Scale bar, 50 μm). F–H Corresponding relative fluorescence intensities of CD11b (F), iNOS (G), and CD206 (H) in the tissues surrounding the implants at 4 and 14 days after surgery. I–K Contents of CXCL2 (I), TNF-α (J), and IL-1β (K) in the soft tissues surrounding the implants on days 4 and 14 after surgery. L Corresponding relative fluorescence intensity of ROS in soft tissues. n = 3 per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are presented as means ± SD.
Immunofluorescence staining was performed to identify the macrophage subtypes in the tissue surrounding the implants. On day 4, both primary skin-resident macrophages (F4/80+ CX3CR1+45,46) and recruited inflammatory macrophages (F4/80+ Ly6C+47) were elevated across all infected groups, with a higher accumulation in the ΔsaeRS strain-infected and fenoprofen-treated groups compared to the WT-infected group. By day 14, the number of both subtypes had significantly decreased in the ΔsaeRS strain-infected and fenoprofen-treated groups, while they continued to increase in the WT-infected group (Supplementary Fig. 15).
Further analysis through immunofluorescence revealed elevated neutrophil infiltration at the infection site on day 4 (Fig. 9C, F). Macrophages in the ΔsaeRS strain-infected and fenoprofen-treated groups exhibited a stronger pro-inflammatory phenotype on day 4, indicated by higher NOS2 expression (Fig. 9D, G). Higher ROS levels were also observed in the tissue surrounding the implants in these groups than in the WT infection group, suggesting robust immune oxidative bactericidal activity at the infection site (Fig. 9E, L). This may explain the reduced bacterial load observed in the later stages of infection. Conversely, on day 14 of infection, as bacterial loads were cleared, macrophages in the ΔsaeRS strain-infected and fenoprofen-treated groups exhibited an anti-inflammatory phenotype with high CD206 expression and significantly reduced ROS levels (Fig. 9D and H), indicating reduced inflammation and enhanced tissue repair capacity. However, macrophages in the WT-infected group still exhibited a strong pro-inflammatory phenotype on day 14, with higher oxidative stress levels in the tissues, suggesting prolonged inflammation and continued tissue damage.
The expression of inflammatory cytokines and chemokines in the tissue surrounding the implant at different time points in the various treatment groups was further assessed (Fig. 9I‒K). The levels of TNF-α, IL-1β, and CXCL2 in the ΔsaeRS strain-infected and fenoprofen-treated groups were higher than those in the WT-infected group on day 4 post-infection and lower on day 14 post-infection. These results suggest that, in the early stages of infection, saeRS mutation or inhibition activates macrophages, causing them to adopt a pro-inflammatory phenotype. Simultaneously, more neutrophils are recruited, which increases ROS levels in the tissue and promotes infection clearance. In contrast, in later stages, as the infection is cleared, macrophages transition to an anti-inflammatory phenotype, leading to reduced expression of inflammatory cytokines and chemokines, decreased presence of neutrophils at the infection site, and lower ROS levels. This is beneficial for tissue repair in the vicinity of the implants. Conversely, in the WT group, activated and recruited macrophages and neutrophils around the implants do not clear the pathogens promptly, resulting in persistent infection. Macrophages remain in a pro-inflammatory state for an extended period and sustained high levels of ROS are present in the tissues. This keeps the tissue in a prolonged state of inflammation and hinders repair and healing processes.
Discussion
Our findings provide novel insights into the role of SaeRS in impairing the immune functions of macrophages during the early stages of infection. Specifically, SaeRS inhibits the production of inflammatory cytokines and chemokines, impairs phagocytosis and bactericidal activities, and decreases cellular oxidative stress. This discovery underscores the significance of SaeRS in the pathogenesis of S. aureus infections. Previous research by Scherr et al.48 reported that during S. aureus biofilm infection, the phagocytic activity of macrophages was suppressed. Given the pivotal role of SaeRS in biofilm formation28,49, SaeRS may impair macrophage functions through the affected biofilm phenotype. Our experimental findings validated this hypothesis. Macrophages exhibit significantly increased invasive and adhesive capabilities towards biofilms formed by the ΔsaeRS strain, and their bactericidal activity against biofilms surpasses that against the WT strain. However, in clinical settings, biofilms tend to develop at the later stage of infection, when adaptive immunity predominates, and macrophages gradually decrease. Although, the SaeRS TCS may modulate macrophage function via biofilm phenotype in laboratory conditions, its significance in clinical settings is limited. Therefore, this study aimed to explore how SaeRS influences macrophages during the early stage of infection, prior to biofilm formation.
Our results indicate that SaeRS compromises macrophage immune functions early on, even before biofilms are established, suggesting additional mechanisms of SaeRS-mediated immune evasion. The formation of bacterial clumping is a common strategy employed by pathogens to evade host immune responses44,50. Specifically, within host environments, bacteria benefit from the abundant extracellular proteins derived from necrotic cells, extracellular matrix components, and damaged tissues. These proteins facilitate bacterial adhesion and aggregation, thereby concealing antigens, which in turn impedes the recognition of bacteria and activation of immune responses44,51. In addition, bacterial clumps exert a significant influence on the overall immune antibacterial effect within the infection site. The clumps protect neighboring smaller targets and enhance the survival ability of nearby planktonic bacteria52. According to our experimental results, the SaeRS TCS has been shown to promote bacterial clumping, likely through the regulation of adhesion-related virulence factors such as FnBPA/B and Efb17. Previous studies have demonstrated that increased expression of FnBPA and FnBPB enhances bacterial clumping, while their loss reduces initial adhesion, suggesting that fibronectin-binding proteins facilitate the initiation phase of biofilm formation and play a crucial role in primary adhesion processes53. Furthermore, research by Ko et al.54 has shown that Efb can bind to fibrinogen, forming a “capsule-like” shield on the surface of the pathogen, which prevents recognition by phagocytic receptors. Based on our study, we speculate that S. aureus utilizes SaeRS TCS-mediated bacterial clustering to mask pathogen-associated molecular patterns (PAMPs), thereby inhibiting the recognition of TLR2 receptors. This mechanism likely hinders the activation of NF-κB in macrophages, which in turn compromises a myriad of immune functions, including the secretion of immune-related cytokines, phagocytosis, bactericidal activities, and ROS production.
To date, the plasticity of macrophages in adopting pro-inflammatory and anti-inflammatory phenotypes has been a focal point in immunological research55,56. During the early stages of infection, macrophages exhibit a pro-inflammatory phenotype. Cells secrete pro-inflammatory and chemotactic factors to recruit additional immune cells to the infected microenvironment57. Immune cells aim to clear the infection through processes such as phagocytosis and ROS generation. Once bacteria are eliminated, macrophages switch to an anti-inflammatory phenotype to promote tissue repair. However, if the infection persists and the microenvironment continues to be invaded by bacteria, the balance between pro-inflammatory and anti-inflammatory states in macrophages is disrupted. Macrophages continue to secrete pro-inflammatory factors and chemokines, leading to prolonged infiltration of inflammatory cells at the infection site and sustained stimulation of ROS, causing severe tissue damage58. Our results emphasize the immunosuppressive role of SaeRS on macrophages during the early stage of infection, which holds significant implications for clinical treatment. The time-dependent accumulation of macrophages at the site of infection, along with the balance between pro-inflammatory and anti-inflammatory phenotypes, profoundly affects the outcome of infection22,59. As observed in our in vivo experiments, compared to the WT strain, the ΔsaeRS inclines macrophages toward a pro-inflammatory phenotype during the early stage of infection. This results in the recruitment of more neutrophils and elevated levels of ROS, which is advantageous for pathogen clearance. Once the infection is eradicated, the macrophages transition to an anti-inflammatory phenotype, characterized by reduced expression of pro-inflammatory factors, decreased neutrophil recruitment at the infection site, and lower ROS levels, thus facilitating the healing of damaged tissue and restoration of homeostasis.
This study has certain limitations. First, while we validated the mechanism involving the NF-κB pathway, which was the most significant altered pathway in our RNAseq analysis, a more thorough examination of other potential pathways influenced by SaeRS is necessary. Second, although we have observed that SaeRS TCS promotes bacterial clumping and impairs macrophage immune functions, which may be related to SaeRS-regulated downstream virulence factors, such as hemolysins, leukotoxins, proteases, and adhesins, the specific virulence factor remains unclear and merits further exploration17.
In summary, this study investigated the impact of the S. aureus SaeRS on the immune functions of macrophages. During the early stage of infection, SaeRS TCS promotes the formation of bacterial clumps, leading to reduced antigen exposure. This, in turn, inhibits TLR2-dependent antigen recognition and NF-kB activation, thereby impairing macrophage immune regulatory functions, as well as phagocytic and bactericidal capabilities against planktonic bacteria. Additionally, the immunosuppressive effects of SaeRS-mediated biofilms on macrophage functions are reconfirmed. These findings highlight the critical role of SaeRS in the pathogenicity of S. aureus throughout infection, underscoring its important clinical implications. Therefore, targeting SaeRS TCS may represent a promising therapeutic strategy to combat S. aureus infections.
Methods
Cell culture
Mouse macrophage RAW264.7 cells were cultured in high-glucose Dulbecco’s Modified Eagle medium (DMEM, Gibco), while human monocyte THP-1 cells were maintained in Roswell Park Memorial Institute Medium 1640 (RPMI 1640, Gibco). Both media were supplemented with 1% penicillin-streptomycin solution (Beyotime, China) and 10% fetal bovine serum (FBS, Gibco), and cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. The THP-1 monocytes were differentiated into macrophages using 100 ng/mL phorbol-12-myristate-13-acetate (PMA, MCE) for 24 h before subsequent experiments.
Bacterial culture and biofilm preparation
The wild-type (WT) Staphylococcus aureus strain ST1792 used in this study was obtained from a patient with periprosthetic joint infection. The saeRS mutant strain (ΔsaeRS) and complement strain (C:ΔsaeRS) strain of ST1792 were constructed by our laboratory. All strains were preserved at -80 °C. Prior to each experiment, strains were cultured on blood agar plates overnight. After that, strains were incubated with tryptic soy broth (TSB) at 37 °C. Subsequently, TSB or PBS was employed to dilute the bacterial solutions to the desired concentration for the following experiments. For the preparation of biofilms, S. aureus suspension (1 × 106 colony units (CFU) mL–1) was seeded in a 24-well plate with TSBG (TSB containing 0.5% glucose) at 37 °C for 24 h. The plate was then washed softly with PBS to remove floating bacteria, leaving biofilms for further experiments.
Co-culture system
RAW264.7 cells and PMA-stimulated THP-1 cells were seeded in plates overnight and co-cultured with different S. aureus strains (MOI = 100), respectively. Meanwhile, another group of WT strain was pretreated with 100 mM fenoprofen calcium dihydrate (Top Science, Shanghai, China, 99.29% purity) overnight to inhibit SaeRS TCS, and then co-cultured with RAW264.7 and THP-1 cells separately. Cells without any treatment were used as the control. To explore the role of the TLR2 receptor in macrophage-SaeRS TCS interaction, macrophages were incubated with 1 μg/mL TLR2 antibody (Bio-Techne, USA) overnight before being co-cultured with S. aureus.
ELISA and RT-PCR Assay
The immunomodulatory effects of macrophages were detected by ELISA and RT-PCR. For the ELISA assay, the cell culture medium was collected and centrifuged. The concentrations of chemokines (CCL3, CXCL2) and inflammatory cytokines (TNF-α, IL-1β, IL-6) were evaluated using ELISA kits (QuantiCyto, China) according to the manufacturer’s instructions.
For RT-PCR assay, the total RNA of macrophages was extracted from different groups using an RNA extraction column kit (EZBioscience, USA). cDNA was then synthesized by a cDNA synthesis kit (EZBioscience, USA) and SYBR Green Master Mix (EZBioscience, USA), and a LightCycler 480 (Roche, USA) was used for quantitative RT-PCR. GAPDH served as the housekeeping gene. All primers utilized are listed in Supplementary Table 1.
Nitric Oxide (NO) Quantification
The amount of nitric oxide (NO) generated by macrophages was determined using the Griess reagent (Beyotime, China). The standard curve was established using commercial NaNO2 (1, 3, 5, 7, and 10 μM). 50 μL of the supernatant from the culture medium of macrophages treated under different conditions was mixed with the Griess reagent to form a diazotization compound. The absorbance was measured at 540 nm.
Antibacterial activity of macrophages in vitro
To access the phagocytosis activity of macrophages, the cells were treated with 200 ug/mL gentamicin for 15 minutes to eliminate extracellular bacteria after co-culture with various groups of GFP-labeled S. aureus for 2 h. After being rinsed three times with sterile PBS, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Subsequently, cells were stained with Actin-Tracker Red 555 (Beyotime, China) for 1 h and DAPI (Beyotime, China) for 15 min. Images were obtained with a DMI8 fluorescence microscope (Leica, Germany). Meanwhile, the phagocytosis rate of macrophages in different groups was detected using flow cytometry.
Spread plate method (SPM) was utilized to further quantify the remaining bacteria within macrophages, allowing for an evaluation of macrophage bactericidal capacity. In brief, after the treatment of 200 μg/mL gentamicin for various durations, cells were permeabilized using 0.1% Triton X-100 to release the intracellular bacteria. Subsequently, after a series of graduated dilutions, 100 μL of the diluted solution was evenly spread onto blood agar plates. The number of bacterial colonies was determined following an 18-hour incubation at 37 °C. Additionally, the intracellular bacterial proliferation within host cells was assessed using SPM.
Detection of biofilm morphology and biomass
As mentioned above, S. aureus strains from different groups were cultured overnight with TSBG at 37 °C for 24 h to form biofilms. The biofilm morphology was observed with a Confocal Laser Scanning Microscope (CLSM) (Leica, Germany). Briefly, biofilms were stained with LIVE/DEAD BacLight Bacterial Viability Kits (Invitrogen, USA) in darkness for 30 min and visualized by a CLSM. The live bacteria emitted green fluorescence.
Crystal violet staining assay was performed to evaluate biofilm biomass. After being washed with sterile PBS, biofilms formed in the 96-well plate were immobilized with 100 μL methanol for 15 min. Then, 0.1% crystal violet was added and stained for 15 min. Finally, the stained biofilms were dissolved in 33% acetic acid and the corresponding absorbance at 590 nm was measured.
Anti-biofilm activity of macrophages in vitro
To access macrophage infiltration into the biofilm, suspensions of GFP-labeled S. aureus strains (1 × 106 CFU mL-1) were seeded into a 24-well plate, forming biofilms in TSBG after different treatments. RAW264.7 and THP-1 cells were stained with CellTracker blue (5 μM). Then, 1 × 105 stained macrophages were added into each well and co-incubated with the biofilms for 2 h at 37 °C. After being washed three times with sterile PBS to remove un-infiltrated macrophages, the biofilms were observed by CLSM (Leica TCS SP8, Germany). SPM was utilized to evaluate bacterial loads within the biofilms before and after co-incubation.
Assessment of macrophage oxidative stress
To measure intracellular ROS levels, the Reactive Oxygen Species Assay Kit (Beyotime, China) was utilized, as described in a previous study60. RAW264.7 and THP-1 (4 × 105 cells well-1) from different groups were plated in 12-well plates. Subsequently, the cells were stained with DCFH-DA (10 μM) in the dark at 37 °C for 30 minutes and further stained with Hoechst 33342 (Beyotime, China) at 37 °C for 20 min. The stained cells were then observed using a DMI8 fluorescence microscope (Leica, Germany). Additionally, the fluorescence intensity was detected using a fluorescence microplate reader (BIO-TEK, USA) with excitation at 485 nm and emission at 530 nm.
For the detection of cellular lipid peroxidation, macrophages were stained with the lipid peroxidation probe C11 BODIPY 581/591 (Glpbio, USA) in the dark for 30 min. The cells were then stained with Hoechst 33342 at 37 °C for 20 min. The stained cells were observed using a DMI8 fluorescence microscope. To access malondialdehyde (MDA) levels, macrophages were lysed using RIPA buffer. After centrifugation, the collected supernatant was used for measuring MDA levels with MDA assay kits (Beyotime, China).
The content of GSH and GSSG were quantified using the GSH and GSSG Assay Kit (Beyotime, China) following the manufacturer’s instructions. Additionally, GSH tracer (MedChemExpress, USA) and Hoechst 33342 were used to stain macrophages, and the resulting staining images were observed using a DMI8 fluorescence microscope.
RNA-sequence analysis
RNA-sequencing was performed by BGI (Shenzhen, China) to analyze the complete transcriptome of macrophages co-cultured separately with WT and ΔsaeRS. All samples were prepared following the instructions provided by BGI. mRNA was enriched using Oligo (dT) magnetic beads, and RNA-seq libraries were constructed. Sequencing was conducted on the MGISEQ2000 platform (BGI, China). The raw data were analyzed using the DESeq2 package. Differential expression of mRNA was identified with cutoffs of |log2 (Fold change) | > 1 and p-value < 0.05.
In addition, a volcano plot was employed to illustrate the differential gene expression. Functional enrichment analysis and pathway enrichment analysis were conducted utilizing the Gene Ontology (GO) database (http://www.geneontology.org) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/). The top 20 enriched GO terms and KEGG pathways (P < 0.05) were visualized through bubble plots. Additionally, Gene Set Enrichment Analysis (GSEA) was performed, and gene sets with a nominal p-value < 0.05 and a false discovery rate (FDR) < 0.25 were considered enriched. All the data processing mentioned above was carried out using the Dr.Tom platform (https://biosys.bgi.com/#/report/login).
Pathway-related Analysis
For western blot assay, macrophages from different groups were lysed with RIPA lysis buffer containing 1% protease and phosphatase inhibitors (Epizyme, China) on ice for 10 minutes. Subsequently, the mixture was centrifugated at 4 °C (14000 rpm, 5 min). Total protein concentration was determined using a BCA protein assay kit (Epizyme, China). Samples containing 10 μg of protein were separated by 10% SDS-PAGE for 50 minutes at 120 V. The separated proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Epizyme, China) for 90 minutes at 220 A. The membranes were blocked with protein-free rapid-blocking buffer (Epizyme, China) for 20 min. After washing three times with TBST buffer, the membranes were incubated overnight at 4 °C with primary antibodies, including p65 (1:1000, Cell Signalling Technology 8242, China), p-p65 (1:1000 Cell Signalling Technology 3033, China) and HRP conjugated GAPDH Antibody(1:1000, ShareBio SB-AB2000, China). The membranes were incubated with the HRP-linked anti-rabbit IgG (1:10000, Cell Signalling Technology 7074, China) at room temperature for 60 minutes. Protein bands were visualized using the Omni-ECL chemiluminescence detection kit (Epizyme SQ101) and a luminescent image analyzer (ImageQuant LAS 4000 mini).
To investigate the nuclear translocation of phosphorylated p65 (p-p65), macrophages from various treatment groups were first fixed and permeabilized. Subsequently, the cells were incubated overnight at 4 °C with a primary p-p65 antibody (Affinity Bioscience, USA). This was followed by a 1-h incubation with FITC-conjugated secondary antibody (Abclonal, China). Afterward, the cells were stained with DAPI for 20 min. Finally, the nuclear translocation of p-p65 was observed using a DMI8 fluorescence microscope.
Animal ethics
All animal surgeries and experiments were approved by the Animal Care and Experiment Committee of Shanghai Sixth People’s Hospital. All experimental procedures followed the NIH Guide for Care and Use of Laboratory Animals guidelines.
Mouse subcutaneous implant infection model
Healthy male BALB/c mice (8 weeks old) were randomly divided into four groups: control, WT, ΔsaeRS, WT+Fen. Anesthesia was induced by intraperitoneal injection of 1% pentobarbital sodium. The dorsal skin of the mice was shaved and disinfected, followed by creating an approximately 1 cm incision using surgical scissors. Sterile titanium plates (d = 1 cm) were then inserted subcutaneously. The incision was sutured, and a prepared bacterial suspension (1 × 106 CFU) was injected onto the surface of the implant. The condition of the infection site, implant retention, and mice weights were recorded on days 0, 1, 4, 7, 10, and 14 post-surgery. In the WT+Fen group, mice received daily intraperitoneal injections of 100 mg/kg fenoprofen, while mice in the other groups were injected with PBS.
On days 4, 7, and 14 after surgery, mice were euthanized and both the implants and surrounding soft tissues were collected. The soft tissues harvested on days 4, 7, and 14 were fixed, dehydrated, embedded with paraffin, and sliced into 4um slices. H&E and Giemsa staining were utilized separately to evaluate the inflammatory response and bacterial burdens. To visually assess the clustering phenotype of S. aureus in infected tissues, immunofluorescence (IF) staining was performed using an S. aureus-specific antibody (Abcam, USA). The stained tissue sections were then observed using a DMI6 microscope (Leica, USA).
To qualify bacterial loads in different groups, on days 4, 7, and 14 after infection, the implants and surrounding soft tissues were ground at high speed to transfer the bacteria into the sterile PBS. Then SPM was employed to measure the bacterial load of implants and surrounding soft tissues in each group.
The microscopic morphology of the biofilm on the implants was observed by scanning electron microscopy (SEM). On days 4, 7, and 14 after surgery, the implants were collected. After being washed with sterile PBS and fixed with 2.5% glutaraldehyde for 8 h at 4 °C, the implants dehydrated in a graded ethanol series (50%, 60%, 70%, 80%, 90%, and 100% v/v) separately for 10 min. Subsequently, the biofilms were freeze-dried overnight and coated with platinum. Finally, SEM (ZEISS GeminiSEM 300, Germany) was used to image the biofilms on the implants.
To evaluate the immunological responses in the tissues around the implants, samples were collected on days 4, 7, and 14 after surgery. ROS staining was performed to assess the levels of oxidative stress within the tissues. Immunohistochemical (IHC) and IF staining were performed to evaluate immune cell infiltration and subtypes over time. For IF, tyramide signal amplification (TSA) technology was employed. Primary iNOS (Abcam, USA), CD206 (Abcam, USA), F4/80 (Abcam, USA), Ly6C (Abcam, USA), CX3CR1 (Abcam, USA), CD11b (Abcam, USA), and MPO (Abcam, USA) antibodies were used with secondary goat anti-rabbit Cy5.5 (Jackson, USA), goat anti-rabbit Cy3 (Jackson, USA) and goat anti-rabbit FITC (Jackson, USA) antibodies, separately. For IHC, slices were incubated with primary F4/80 (Abcam, USA) and Ly6G (Abcam, USA) antibodies followed by secondary antibody peroxidase-conjugated affinipure goat anti-rabbit IgG (H + L) (Jackson, USA). All stained slices were imaged using an optical microscope or a DMI6 microscope. Finally, ELISA was conducted to measure the levels of inflammatory cytokines and chemokines, including TNFα, IL-1β, and CXCL2 (QuantiCyto, China), in the tissues surrounding the implants on days 4 and 14 after surgery.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8 software. All results were presented as means ± standard deviation. Data were analyzed using one-way ANOVA and T-test to determine statistical significance, with the threshold for significance set at P < 0.05. Noteworthy labels and annotations were integrated into the associated studies for comprehensive clarity.
Data availability
Data will be made available on request.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 82272511), the experimental animal study support project of the Shanghai Science and Technology Commission (Grant No. 21140904800), the Undergraduate Training Program on Innovation, Shanghai Jiao Tong University School of Medicine (Grant No. 1723Y039).
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M.L.: Formal analysis, Investigation, Writing—original draft. B.W.: Data curation, Formal analysis, Investigation, Writing—review & editing. J.C.: Data curation, Methodology, Writing—original draft. L.J.: Formal analysis, Investigation. Y.Z.: Formal analysis, Methodology. G.G.: Data curation, Visualization. F.J.: Investigation, Methodology. Y.H.: Formal analysis. C.W.: Methodology, Visualization. Y.Y.: Formal analysis, Investigation. J.T.: Investigation, Visualization. P.H.: Conceptualization, Writing—review & editing. J.Y.: Conceptualization, Formal analysis, Investigation, Writing—review & editing. H.S.: Conceptualization, Project administration, Funding acquisition, Writing—review & editing.
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Li, M., Wang, B., Chen, J. et al. Staphylococcus aureus SaeRS impairs macrophage immune functions through bacterial clumps formation in the early stage of infection. npj Biofilms Microbiomes 10, 102 (2024). https://doi.org/10.1038/s41522-024-00576-8
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DOI: https://doi.org/10.1038/s41522-024-00576-8
