This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.
Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment.
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.
Staphylococcus aureus is both a life-threatening pathogen and a commensal bacterium of the skin and the mucosa that colonizes two billion individuals around the world1. In humans, nasal carriers of S. aureus have an increased risk of infection with their own strain of carriage; however, the multifactorial determinants of S. aureus mucosal carriage are still unclear1,2. In addition to acute infections, patients can also develop chronic S. aureus infections that are often challenging to cure3. A better understanding of host-pathogen interactions during colonization and infection is crucial for developing novel therapeutic strategies and improving patient management.
In vitro, S. aureus can trigger its internalization into host cells expressing the α5β1 integrin4. The tripartite interaction between the staphylococcal fibronectin-binding proteins anchored to the cell wall of S. aureus, the fibronectin, and the β1 integrin expressed at the host cell surface is well known as the main pathway of S. aureus internalization in NPPCs such as keratinocytes, osteoblasts, fibroblasts, and epithelial and endothelial cells4. Recent studies show that S. aureus can be found inside human cells during nasal colonization5,6 and infection7. However, the role of the intracellular reservoir in the pathogenesis of S. aureus infection remains unclear. The host cells could act as a shelter for S. aureus, which is protected from both the immune system8 and most antimicrobial compounds6,9.
The lysostaphin protection assay, described by Proctor10 earlier in the 1980s, enables the study of bacterial and host factors involved in the internalization of S. aureus isolates. Lysostaphin is a bacteriocin produced by Staphylococcus simulans, which exhibits potent activity against almost all S. aureus isolates, including antibiotic-resistant strains11. Lysostaphin has been used to destroy only extracellular S. aureus to enable the counting of only viable intracellular bacteria12. This technique has been widely used and has contributed to the discovery of several virulence factors of S. aureus. Gentamycin, alone and combined with lysostaphin, is also widely used to study intracellular bacteria.
However, a recent study showed that gentamycin enters eukaryotic cells and reaches internalized bacteria in a time- and concentration-dependent manner13. This study also demonstrated that lysostaphin does not enter eukaryotic cells, confirming that a lysostaphin-based enzyme protection assay (EPA) is the most accurate assay for quantifying intracellular S. aureus load by culture13. Regardless of which compound is used to destroy extracellular bacteria (e.g., lysostaphin or gentamycin), it should be removed by washing the cells before plating intracellular S. aureus on agar plates. Successive washes may result in the detachment of cells, especially poorly adherent cells (e.g., heavily infected cells), which would lead to an underestimation of the intracellular S. aureus load. This paper describes in detail how EPA can be used to quantify the intracellular S. aureus load and to measure the intracellular efficacy of antimicrobials compounds using an in vitro model. Of note, a simple method has been proposed to improve the reliability of intracellular load quantification by avoiding intensive washes.
1. Culture of human epithelial cells
2. Culture of S. aureus strains
3. Infection assay with S. aureus
The results of S. aureus internalization by A549 epithelial cells are depicted in Figure 1A. A549 cells were inoculated with S. aureus SF8300 WT and SF8300 ΔfnbA/B, which lacks fibronectin-binding proteins A and B, at an MOI of 1 for 2 h. To destroy extracellular S. aureus, lysostaphin was added to the culture medium, and the cells were incubated for 1 h. Next, lysostaphin was either removed by washing for EPA or inactivated with proteinase K for iEPA. Then, the cells were disrupted in lysis buffer, and the bacterial load was quantified by culture. By using EPA, the mean intracellular loads were 4.46 and 0.49 Log CFU/mL for SF8300 WT and SF8300 ΔfnbA/B, respectively (Figure 1A, green bars). Using iEPA, the mean intracellular loads were 4.53 and 0.56 Log CFU/mL for SF8300 WT and SF8300 ΔfnbA/B, respectively (Figure 1A, red bars). It is interesting to note that both EPA and iEPA showed similar results, which can be explained by the ease of performing the washes when the cells are in good condition and because the S. aureus-induced cytotoxicity is very low in these experimental settings (data not shown).
The results of intracellular activity of vancomycin, rifampicin, and levofloxacin against S. aureus are depicted in Figure 1B. To measure the intracellular activity of these antibiotics, HaCaT cells were inoculated with S. aureus ATCC 29213 at an MOI of 1 for 2 h. The cells were incubated with lysostaphin, with or without the antimicrobial compounds to be tested, for 24 h. Next, lysostaphin and the antimicrobial compounds were removed by washing. The cells were disrupted in lysis buffer, and the bacterial load was quantified by culture. The mean intracellular loads were 4.57, 4.51, 3.03, and 2.91 log CFU/mL for control, vancomycin (50 µg/mL), rifampicin (7 µg/mL), and levofloxacin (10 µg/mL), respectively (Figure 1B).
Figure 1: Intracellular Staphylococcus aureus load in epithelial cells. (A) Enzyme protection assay (green bars) and improved enzyme protection assay (red bars) in A549 cells infected with S. aureus SF8300 WT and ΔfnbA/B. (B) Intracellular activity of antimicrobial compounds in HaCaT cells infected with S. aureus ATCC 29213. Bars represent the mean values of three independent experiments performed in triplicate. Error bars represent the standard deviations. **** p < 0.0001. Abbreviations: Ctrl = control; cfu = colony-forming units. Please click here to view a larger version of this figure.
The assays described here are valuable for studying the extent of internalization and the intracellular survival of S. aureus in NPPCs, as well as the intracellular efficacy of antimicrobial compounds6,15,16. Some steps in both assay protocols can be critical. The health condition and the density of the cells must be perfectly controlled and consistent between independent experiments. The bacterial inoculum must be carefully standardized to obtain a real MOI close to the targeted theoretical MOI. In general, care must be taken not to detach any of the cells while pipetting. The washes to remove lysostaphin and antibiotics are critical steps in the EPA. The use of proteinase K has been found to improve this step when no antibiotic is used (see below). Last but not least, the cells should be fully detached in each well and thoroughly homogenized after the incubation with the lysis buffer to reliably quantify the S. aureus intracellular load.
In some instances, issues may be encountered, and several points must be checked first. In case of a lack of reproducibility, it must be kept in mind that S. aureus can form clumps, making quantification by absorbance inaccurate. The clumping of bacteria can be increased by centrifugation and washing steps if the culture medium is to be replaced (e.g., for eliminating a secreted protein). The bacterial suspension should be used rapidly because bacteria continue to grow at room temperature. The lysostaphin efficacy could decrease because of incorrect storage conditions, suboptimal pH for enzyme activity in the culture media, variability in the enzymatic activity between batches and providers, and lack of lysostaphin sensitivity of some strains in specific growth conditions. Phenol red could have a slight bacteriostatic effect, especially when the culture medium is relatively poor in nutrients compared to the typical broths used for growing bacteria. Thus, it is advisable to use a cell culture medium without phenol red, which also improves fluorescence microscopic observations by reducing the background noise.
Although this method is a valuable tool to study the intracellular fate of different strains, some limits of the method should be considered. The use of a very high MOI can overload the capability of internalization by NPPCs and level out the differences between the different strains tested. The extent of internalization of the most cytotoxic strains may be underestimated because lysostaphin (or antibiotics) rapidly destroys S. aureus that is released by damaged cells. Thus, experiments with extended durations (i.e., to study intracellular survival or intracellular activity of antibiotics) are easier to set up with strains with low cytotoxicity. Therefore, the incubation time and the MOI should be accurately adjusted according to the strain virulence, the cell type, and the experimental aim.
The method described here with the use of lysostaphin is more reliable than those based on gentamicin because, unlike lysostaphin, gentamicin tends to be internalized by host cells13. The other advantage is the possibility to inactivate the lysostaphin. Inhibition of lysostaphin activity was reported by Kim et al.13 with the use of EDTA to chelate zinc ions or 1,10-phenanthroline; however, intensive washes are still required to remove the enzyme before plating of the bacteria. Here, proteinase K enables rapid inactivation of lysostaphin. We observed that cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. By skipping the final washing step, the iEPA method greatly simplified technical handling and enabled the recovery of the internalized bacteria in loosely adherent or already detached cells.
The more concentrated reagents and buffers used in iEPA also helped reduce pipetting effort and minimize the loss of cells. In addition, iEPA can be used with cells in suspension, as well as with organoids that are difficult to wash. In conclusion, enzyme protection assays enable the study of the extent of internalization and the intracellular fate of S. aureus, as well as the intracellular activity of antimicrobials drugs with different in vitro models. Improvements should be made to better characterize the relationship between internalization and cytotoxicity to better appreciate the importance of developing drugs capable of reaching S. aureus inside the cell.
The authors have nothing to disclose.
S. aureus strains SF8300 WT and SF8300 ΔfnbA/B were generously gifted by Prof. Binh Diep (University of California, San Francisco, USA). This work was supported by a grant of the FINOVI association (#AO13 FINOVI) under the aegis of the Foundation for the University of Lyon.
24-well plate | CORNING-FALCON | 353047 | |
A549 cell line | ATCC | CCL-185 | |
Acetate buffer solution pH 4.6 | Fluka | 31048 | Used to prepare lysostpahine stock solution at 10 mg/mL in 20 mM sodium acetate. |
AMBICIN (Recombinant lysostaphin) | AMBI | LSPN-50 | Lyophilized recominant lysostaphin. Freeze at -80 °C for long-term storage. |
COS – Colombia agar + 5% sheep blood | Biomerieux | 43049 | Any agar plate suitable for growing staphylococci can be used instead. |
Densitometer WPA CO8000 | Biochrom Ltd. | 80-3000-45 | Cell density meter |
Dulbecco’s Modified Eagle’s Medium, high glucose with phenol red | Sigma-Aldrich | D6429 | |
Dulbecco’s Modified Eagle’s Medium, high glucose without phenol red | Sigma-Aldrich | D1145 | |
Dulbecco’s Phosphate Buffered Saline | Sigma-Aldrich | D8537 | |
Dulbecco′s Phosphate-buffered Saline with MgCl2 and CaCl2, sterile-filtered | Sigma-Aldrich | D8662 | |
Dulbecco′s Phosphate-buffered Saline, sterile-filtered | Sigma-Aldrich | D8537 | |
Easyspiral dilute | Interscience | 414000 | Automatic diluter and spiral plater |
Fetal bovine serum | Gibco | 10270-106 | |
HaCaT cell line | Cell lines service (CLS) | 300493 | |
Hoechst 33342, Trihydrochloride, Trihydrate, 10 mg/mL Solution in Water | Fisher scientific | 11534886 | |
Propidium iodide, 1.0 mg/mL solution in water | Invitrogen | P3566 | |
Proteinase K, recombinant 20 mg/mL | Eurobio | GEXPRK01-B5 | > 30 U/mg, lot 901727 |
Scan 4000 | Interscience | 438000 | Automatic colony counter |
Sterile water | OTEC | 600500 | |
T-75 culture flask | CORNING-FALCON | 353136 | |
TC20 Automated cell counter | Biorad | 1450102 | Automatic cell counter |
Tris 1 M pH 8.0 | Invitrogen | AM9855G | Used to prepare lysostaphine working solution at 1 mg/mL in 0.1 M Tris-HCl. |
Triton X-100 | Sigma-Aldrich | T8787 | |
Trypsin – EDTA solution | Sigma-Aldrich | T3924 | 0.05% porcine trypsin and 0.02% EDTA in Hanks′ Balanced Salt Solution with phenol red |
Wide-field fluorescence microscope | Nikon | Ti2 |