Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds
September 8th, 2021
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.
The enzyme protection assay enable to study the extent of Staph aureus internalization and its ability to survive inside adherent cells, as well as the efficacy of antimicrobial compound. The improved enzyme protection assay greatly simplifies technical handling, and enable to recover internalized bacteria from weakly-adherent cells and cells in suspension. Demonstrating the procedure will be Josselin Rigaill, medical doctor and PhD student from my laboratory.
Two days before the infection assay, seed A549 epithelial cells at a density of 2.0 times 10 to the fifth cells per milliliter per well of a 24-well plate and incubate the cells for 48 hours at 36 degrees Celsius in 5%carbon dioxide, until they reach 100%confluence. On the day of the assay, remove and discard the spent culture medium from the three wells dedicated for counting the A549 cells, and add one milliliter of complete infection medium containing five micrograms per milliliter of Hoechst 33342 and one microgram per milliliter of propidium iodide. Incubate the cells for 30 minutes, then, using a wide-field fluorescence microscope, count the number of cells and calculate the cell viability.
To prepare the bacterial suspension, dispense 25 milliliters of complete infection medium in a tube, and prewarm it at 36 degrees Celsius. Next, adjust the Staphylococcus aureus suspension to an OD of 0.5 in a complete infection medium using a cell density meter, then prepare 20 milliliters of bacterial suspension for cell inoculation by diluting the culture in a complete infection medium to achieve an MOI of one according to the number of cells per well. Next, using an automatic spiral plater, determine the Staphylococcus aureus load of the diluted bacterial suspension to be used for the cell inoculation step, and incubate the agar plates for 18 to 24 hours at 36 degrees Celsius.
The next day, count the number of colonies with a colony counter to calculate the accurate MOI for each strain tested. Before cell inoculation, observe every well of the 24-well plate by low magnification microscopy to ensure that the cells are healthy and growing as expected, then remove and discard the spent cell culture medium from the 24-well plate and add 500 microliters of the bacterial suspension to each well containing 100%confluent cells. Incubate the cells for two hours at 36 degrees Celsius in 5%carbon dioxide.
To quantify the intracellular bacteria with the improved enzyme protection assay, prepare 4x lysis buffer, 2%Triton X-100, trypsin-EDTA, and lysostaphin stock in working solutions as mentioned in the text manuscript. Next, prepare 6.25 milliliters of complete infection medium supplemented with lysostaphin by adding six milliliters of complete infection medium to 250 microliters of the lysostaphin working solution. Then, add 250 microliters of the complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swiveling the plate by hand.
To allow the lysostaphin to kill the extracellular bacteria, incubate the cells for one hour at 36 degrees Celsius in 5%carbon dioxide, then inactivate the lysostaphin by adding 10 microliters of proteinase K at 20 micrograms per milliliter into each well and incubating the cells for two minutes at room temperature. Next, add 250 microliters of 4x lysis buffer to lyse the cells by osmotic shock, and incubate the cells for 10 minutes at 36 degrees Celsius. After the incubation, pipette the cell lysate up and down several times to ensure that the cells are fully lysed and homogenized, then use an automatic spiral plater to determine the Staphylococcus aureus load of each well and incubate the agar plates for 18 to 24 hours at 36 degrees Celsius.
The next day, count the number of colonies with a colony counter to calculate the intracellular Staphylococcus aureus load of each well. The internalization of two strains of Staphylococcus aureus by A549 epithelial cells is depicted here. Using the enzyme protection assay that includes washes to remove lysostaphin, the mean intracellular loads were 4.46 and 0.49 log cfu per milliliter for the SF8300 wild type and the isogenic mutant lacking the fnbA and B genes, respectively.
Using the improved enzyme protection assay that uses proteinase K to inactivate lysostaphin, the loads were 4.53 and 0.56 log cfu per milliliter, respectively. Intracellular activity of vancomycin, rifampicin, and levofloxacin against Staphylococcus aureus measured using enzyme protection assay is depicted here. The mean intracellular loads were 4.57 log cfu per milliliter for control, 4.51 for vancomycin, 3.03 for rifampicin and 2.91 for levofloxacin.
Intensive washes to remove the enzyme tend to detach the most infected cells, which can give inaccurate results and support the use of the improved protocol. The improved enzyme protection assay make it easier to study Staph aureus internalization with organoid, and can be easily adapted on high content screening system that's used by putting
Overview
This study focuses on understanding the internalization of Staphylococcus aureus in human host cells and its survival mechanisms within those cells. Additionally, it evaluates the effectiveness of various antimicrobial compounds against this bacterium using an enhanced enzyme protection assay.
Key Study Components
Research Area
- Microbial pathogenesis
- Antimicrobial response
- Cellular internalization dynamics
Background
- Staphylococcus aureus poses significant clinical challenges due to its ability to survive within host cells.
- Understanding its internalization can inform treatment strategies against infections.
- Previous methods may have limitations in accurately quantifying bacterial internalization.
Methods Used
- Improved enzyme protection assay for studying bacterial internalization
- A549 epithelial cells for host cell model
- Fluorescence microscopy, lysostaphin treatment, and colony counting techniques
Main Results
- Mean intracellular loads of Staphylococcus aureus were significantly quantified using the improved assay.
- Varied efficacy of antimicrobial compounds, with vancomycin showing higher intracellular activity.
- The modified protocol facilitated more accurate measurement of bacterial internalization.
Conclusions
- The study presents an effective method for quantifying Staphylococcus aureus survival in host cells.
- This research offers insights vital for developing treatment strategies against bacterial infections.
Frequently Asked Questions
Executive Industry Relevance
Accurate quantification of intracellular bacterial load is critical for evaluating antimicrobial efficacy in non-professional phagocytic cells, where traditional assays may underestimate pathogen persistence. The improved enzyme protection assay addresses technical limitations in recovering internalized bacteria from weakly adherent or suspended cells, enhancing reliability in early-stage antimicrobial screening. This supports mechanistic de-risking by enabling consistent measurement of compound activity against intracellular Staphylococcus aureus, informing lead optimization decisions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of virulence factor contributions to internalization and intracellular survival, supporting target validation for anti-infective strategies.
- Operational Value: Provides a reproducible method to compare isogenic strains, clarifying genetic determinants of pathogen persistence.
Screening & Assay Development
- Scientific Value: Generates quantitative intracellular CFU readouts to assess compound efficacy against internalized Staphylococcus aureus.
- Operational Value: Eliminates washing steps that cause cell detachment, improving assay robustness in adherent and suspension models including organoids.
- Scientific Value: Facilitates high-content screening adaptation by simplifying lysis and recovery steps, increasing throughput for antimicrobial profiling.
Translational & Preclinical Research
- Scientific Value: Supports disease-relevant modeling using adherent non-professional phagocytic cells and 3D organoid systems to mimic intracellular niches.
- Operational Value: Enables continuity from hit identification to preclinical evaluation by providing a standardized intracellular bacterial burden metric.
Pipeline & Workflow Integration
The assay fits within the antimicrobial discovery continuum, linking target validation through phenotypic screening to preclinical efficacy assessment by quantifying intracellular pathogen load as a key pharmacodynamic endpoint.
- Discovery Biology: Supports hypothesis testing of host-pathogen interactions and virulence mechanisms via controlled MOI and lysostaphin protection steps.
- Screening: Delivers reproducible, quantitative intracellular CFU measurements that enable comparison of antimicrobial compounds across conditions.
- Analytics: Generates log CFU/mL readouts that allow teams to rank compound intracellular activity and assess time-kill or post-infection treatment effects.
- Translational Research: Connects to preclinical workflows by providing a translatable intracellular efficacy metric applicable to primary cells and organoid models.
- Enterprise Reuse: Establishes a reusable intracellular infection platform adaptable to multiple pathogens and cell types beyond Staphylococcus aureus.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in antimicrobial action by distinguishing extracellular from intracellular effects.
- Operational Value: Standardizes intracellular bacterial recovery, minimizing variability due to cell loss during processing.
- Strategic Value: Improves go/no-go decisions by revealing compounds with true intracellular activity, reducing late-stage failure risk.
- Portfolio Impact: Enables risk-adjusted prioritization of leads based on intracellular efficacy data, improving capital efficiency in anti-infective programs.
Implementation Considerations
- Requires expertise in cell culture, multiplicity of infection calculation, and anaerobic or facultative pathogen handling.
- Depends on access to lysostaphin, proteinase K, lysis buffers, and spiral platers for accurate CFU quantification.
- Necessitates standardization of MOI, lysostaphin exposure time, and inactivation protocols across laboratories.
- Adaptation to primary cells or 3D models may require optimization of lysostaphin penetration and lysis conditions.
- Practical limitation: Assay duration exceeds 24 hours, which may constrain ultra-high-throughput screening campaigns.
Biopharma FAQs
Chapters in this video
0:05
Introduction
0:41
Determination of Cell Density and Viability
1:33
Preparation of the Bacterial Suspension
2:32
Cell Inoculation
3:03
Quantification of Intracellular Bacteria with Improved Enzyme Protection Assay (iEPA)
4:44
Results: Intracellular Staphylococcus aureus Load in Epithelial Cells
5:54
Conclusion
