Staphylococcus aureus (S. aureus) has the capability to disseminate throughout the body, causing persistent and recurrent infections. To better understand these processes, this study establishes an intracellular infection model for S. aureus. This model will provide a crucial foundation for investigating the mechanisms behind intracellular infections.
S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.
S. aureus is a highly contagious pathogen that can cause a range of infections, including skin and soft tissue infections, sepsis, meningitis, pneumonia, and endocarditis1. The clinical misuse of antibiotics has led to increased resistance in S. aureus and the emergence of methicillin-resistant Staphylococcus aureus (MRSA), which poses a significant public health threat in many countries2.
Although S. aureus is not traditionally classified as an intracellular pathogen, emerging evidence suggests that it can persistently colonize host cells following invasion3. The ability of S. aureus to survive intracellularly within host phagocytes is increasingly recognized as a mechanism that facilitates metastatic infection and dissemination throughout the host4,5,6. S. aureus secretes various virulence factors, which create an immune environment that promotes its survival and complicates the host's ability to fully eliminate it7. The accessory gene regulator (agr) and Staphylococcal helper elements (Sae) are two important virulence regulators that are closely related to the survival of Staphylococcus aureus in phagocytes8,9. The agr system is a quorum-sensing mechanism that regulates the expression of numerous virulence factors in S. aureus. It controls the production of toxins and other factors that facilitate bacterial survival and dissemination. During intracellular infection, the agr system plays a critical role in the regulation of virulence factors that are essential for the bacterium's ability to evade host immune responses and survive within host cells. Studies have shown that the agr system influences the bacterium's ability to escape from phagosomes and persist within macrophages. The absence of agr can lead to reduced bacterial survival within host cells and decreased virulence10,11. The Sae system is a two-component regulatory system that controls the expression of several virulence factors in S. aureus. It is involved in the regulation of toxins and enzymes that contribute to the bacterium's ability to invade and damage host tissues. The Sae system also plays a crucial role in S. aureus intracellular survival. It influences the bacterium's ability to resist killing by host phagocytes and evade autophagic degradation12,13.
When pathogens invade, macrophages have phagocytic functions, which can engulf and kill foreign pathogens and activate adaptive immune response14. Most invading bacteria are phagocytosed by macrophages, which then activate various killing mechanisms to eliminate them. However, some S. aureus bacteria can survive within macrophages, leading to persistent infection of the host. In addition to bacterial proteins, the host also impacts the survival and proliferation of S. aureus within macrophages by secreting cytokines15,16,17. Some studies indicate that S. aureus can evade degradation by residing in autophagosomes, creating an intracellular niche that promotes dissemination18. S. aureus escapes autophagic degradation by blocking autophagy flux (e.g., LC3-II, p62) and increasing pH within autolysosomes after macrophage invasion19. This immune evasion is achieved through the regulation of S. aureus virulence factors and autophagy, leading to persistent, hidden infections.
Clearing intracellular infections of S. aureus is crucial for managing persistent and latent infections in clinical practice. Currently, antibiotics are the primary treatment for S. aureus infections, with vancomycin serving as the last line of defense for MRSA infections20,21. However, numerous studies have shown that existing antibiotics are ineffective at eliminating intracellular S. aureus, both invivo and in vitro22,23,24.
There is currently no unified standard for the various intracellular infection models of S. aureus25,26,27, as the conditions of each model differ significantly. Consequently, the same criteria cannot be applied to assess the effectiveness of these models. In this study, we established a universal intracellular infection model of Staphylococcus aureus by optimizing the experimental conditions. This model offers greater convenience compared to others, as it allows for the initial infection of bacteria into cells in vitro, followed by the delivery of these infected cells into the body.
To better understand the mechanisms of intracellular S. aureus infection and develop related drugs, we established both in vitro and in vivo models. A stable intracellular infection model was successfully created in vitro by infecting RAW264.7 and co-culturing them with antibiotics. Then, peritoneal macrophages were extracted and formed into intracellular infections. An intracellular infection model in mice was established by injecting these peritoneal macrophages.
Experimental animals, 6-8 weeks old specific pathogen-free (SPF) female BALB/c mice, were purchased from the Beijing HFK Bioscience Co., Ltd (Beijing, China). All animal studies were approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University and were performed in accordance with the institutional and national policies and guidelines for the use of laboratory animals. The mice were kept and vaccinated in SPF facilities and provided free access to sterile food and water. Animals were randomly divided into groups and conceded an adaptation time of at least 7 days before the beginning of the experiments.
1. Preparation for S. aureus
2. Establishment of intracellular infection model in vitro
3. Establishment of in vivo intracellular infection model
Intracellular infection models of S. aureus were successfully established both in vitro and in vivo. By optimizing the experimental conditions for phagocytosis and extending both the concentration and duration of antibiotic treatment, some S. aureus survived within the macrophages (Figure 1). To further assess the antibiotic resistance of S. aureus, macrophages infected with the MRSA252 were treated with antibiotics for 2 h until the cell supernatant no longer contained S. aureus, while some bacteria persisted within the macrophages (Figure 2A). A confocal laser scanning microscope revealed that antibiotics could not kill S. aureus within the cells (Figure 2B). Additionally, bacterial colonization assays in mice showed that while vancomycin could eliminate planktonic S. aureus, it was ineffective against bacteria residing inside cells (Figure 5B). Thus, the intracellular infection model of S. aureus has been successfully established.
Figure 1: Optimal experimental conditions for intracellular infection of MRSA252 in peritoneal macrophages. (A) Bacterial load in peritoneal macrophages infected with MRSA252 at different MOI values. (B) Bacterial load in peritoneal macrophages infected with MRSA252 and treated with varying concentrations of gentamicin. (C) Bacterial load in peritoneal macrophages infected with MRSA252 after treatment with 100 µg/mL gentamicin for different durations. Data are expressed as mean ± SEM (n=3), and one-way ANOVA with Dunnett's post-hoc test was used to determine significance (**** p < 0.0001, ** p < 0.01, * p < 0.05). Please click here to view a larger version of this figure.
Figure 2: Intracellular S. aureus resists antibiotic treatment. (A) Image of bacterial plate coating of cell suspension and cytoplasm. (B) Confocal microscopy showed the growth of bacteria in RAW264.7 (red) infected by MRSA252 (green) at 4 h points in the presence of antibiotics (scale bar = 10 µm). Please click here to view a larger version of this figure.
Figure 3: Procedure for intraperitoneal injection. (A) Grasp the mouse securely. (B) Identify the injection site for the intraperitoneal injection. Please click here to view a larger version of this figure.
Figure 4: Procedure for peritoneal macrophage extraction. (A) Cut the surface skin of the abdomen. (B) Perform blunt separation of the skin. (C) Skin separation exposes the peritoneum. (D) Lavage the abdominal cavity. (E) Collect and further lavage the abdominal cavity. Please click here to view a larger version of this figure.
Figure 5: Intracellular infection with MRSA252 is resistant to vancomycin killing. (A) Graphical abstract illustrating the experimental design for establishing infection models with planktonic versus intracellular bacteria. The graphical abstract was drawn by Figdraw. (B) Bacterial loads in the kidney: Bacterial loads were detected 1 day after infection. CFU, colony-forming units. Vanco represents vancomycin. Data were expressed as mean ±SEM (n=5 biologically independent samples), and one-way ANOVA with Dunnett's post-hoc test was used to determine significance (****p < 0.0001, ns represents no significance). Please click here to view a larger version of this figure.
S. aureus, as a facultative intracellular pathogen, can invade and survive in various cell types, using this capability to evade antibiotics and immune responses during infection30. This study established an intracellular infection model of S. aureusin vivo to provide a foundation for investigating the pathogen's intracellular infection mechanisms. By exploring the impact of various MOI values on macrophage phagocytosis of S. aureus, as well as the efficacy of different antibiotic concentrations and treatment durations, the optimal conditions for establishing an intracellular infection model were determined (Figure 1).
When constructing the in vitro model of S. aureus intracellular infection, adjust the S. aureus at the logarithmic growth phase to the desired concentration and add it to the cell culture plate. Ensure that the medium does not contain antibiotics, as their presence can hinder the pathogen's ability to enter the cells. After 2 h of infection, a medium containing 100 µg/mL concentration of gentamicin was added to treat the infected cells. Cell colonization experiments (Figure 2A) demonstrated that gentamicin effectively eliminated extracellular bacteria while the remaining bacteria were internalized by the cells. Confocal microscopy further confirmed that this approach facilitated the entry of bacteria into cells, establishing an intracellular infection (Figure 2B).
In this study, mice were stimulated with starch broth to produce peritoneal macrophages, which were then extracted and infected with MRSA252 in vitro. These infected peritoneal macrophages were subsequently injected into mice. For control, mice were also injected with 3 x 106 CFU of planktonic MRSA252. After these injections, vancomycin treatment was administered to all mice.
Vancomycin was administered to mice 24 h prior to assessing bacterial levels, establishing a critical timeframe for intracellular bacterial infection. In mice treated with planktonic MRSA252, vancomycin nearly eradicated the bacterial load in the kidneys. However, the intracellular infection group still exhibited significant bacterial presence despite receiving a lower challenge dose of 1.8 x 106 CFU compared to the planktonic group (Figure 5B). Thus, we conclude that the model was successfully established. In comparison to other models25, we first stimulated mouse peritoneal macrophages for in vitro infection, resulting in an increased recovery of these cells.
In summary, the bacterial invasion and colonization of cells can be observed more directly using MRSA252 engineered with green fluorescent protein. The strain can also be used to evaluate the interaction between S. aureus and the host, for example, the invasion and adhesion of S. aureus to the host. Additionally, the model uses conditions from in vitro intracellular infection to refine and optimize the in vivo model. However, it does not fully explore bacterial colonization in other organs post-intracellular infection and may not entirely reflect clinical infection scenarios. A more in-depth investigation of intracellular infection patterns could improve the model's simulation of clinical situations.
The bacterial invasion and colonization of cells can be directly observed using MRSA252 engineered with green fluorescent protein. This strain also facilitates the evaluation of interactions between S. aureus and the host, including the invasion and adhesion processes. Additionally, the model leverages conditions from in vitro intracellular infection to refine the in vivo model. However, it falls short of fully exploring bacterial colonization in other organs post-intracellular infection in this work and may not accurately represent clinical infection scenarios. A more in-depth investigation of intracellular infection patterns is needed to enhance the model's simulation of clinical situations.
The authors declare that they have no competing interests.
This work was supported by the National Natural Science Foundation of China (NSFC, Grant No.32300779, NO.32270989), Natural Science Foundation of Chongqing (CSTB2022NSCQ-MSX0156), Science and Technology Research Project of Chongqing Education Commission (KJQN202312802) and China Postdoctoral Science Foundation (2024M754250).
Name | Company | Catalog Number | Comments |
24-well plate | Corning Incorporated, USA | 3524 | |
4 % paraformaldehyde solutione | BBI, UK | E672002-0500 | |
6-well plate | Corning Incorporated, USA | 3516 | |
Beef extract powder | BBI, UK | A600114-0500 | |
Biohazard safety equipment | Heal force, China | VS-1300L-u | |
Cell incubator | ESCO, Singapore | CCL-170B-8 | |
Cell scraper | Nest | 710001 | |
Centrifuge M1416R | RWD, China | M1416R | |
Centrifuge tube | Guanghou Labselect, China | CT-002-50A | |
Confocal laser scanning microscope (CLSM) | Zeiss, Germany | 880 | |
Confocal petri dish | Biosharp, China | BS-20-GJM | |
DAPI dye | Shanghai Beyotime, China | C1006 | |
DIL working fluid | Shanghai Beyotime, China | C1991S | |
Dulbecco’s Modified Eagle Medium | Thermo Gibco, USA | C11995500BT | |
Fetal Bovine Serum | Hyclone | SV30208.02 | |
Gentamycin | Shanghai Sangon, China | B540724-0010 | |
Incubator | Shanghai Hengzi, China | HDPF-150 | |
Lysozyme | Beijing Solarbio, China | L9070 | |
MRSA252 | Third Military Medical University, China | null | |
MRSA252(GPF) | Third Military Medical University, China | null | |
Penicillin and Streptomycin | Shanghai Beyotime, China | C0222 | |
Phosphate Buffer Solution | Shanghai Beyotime, China | ST476 | |
Saline | Sichuan Kelun, China | null | |
Sodium chloride | Shanghai Macklin, China | S805275 | |
Starch soluble | Shanghai Sangon, China | A500904-0500 | |
Triton X-100 | Shanghai Beyotime, China | P0096-100ml | |
Tryptic Soy Agar (TSA) plates | Beijing AOBOX Biotechnology Co., LTD,China | 02-130 | |
Tryptic Soy Broth (TSB) medium | Beijing AOBOX Biotechnology Co., LTD,China | 02-102K | |
Tryptone | OXOID, UK | LP0042B | |
Vancomycin | Shanghai Beyotime, China | ST2807-250mg | |
RAW264.7 cell | USA, ATCC | null |
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