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Updated: Dec 30, 2025
Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes
Published on: January 3, 2020
Quantifying the Cytotoxicity of Staphyloccus aureus Against Human Polymorphonuclear Leukocytes
Jennifer G Dankoff1, Kyler B Pallister1, Fermin E Guerra1
1Department of Microbiology and Immunology, Montana State University.
Abstract:
Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.
Insights
This study details methods to purify human neutrophils and quantify Staphylococcus aureus cytotoxicity. Researchers assess the disruption of neutrophil membrane integrity by S. aureus leukocidins.
Area of Science:
- Microbiology
- Immunology
- Cell Biology
Background:
- Staphylococcus aureus produces leukocidins that compromise polymorphonuclear leukocyte (PMN) membrane integrity.
- Understanding host-pathogen interactions is crucial for combating bacterial infections.
Purpose of the Study:
- To describe a protocol for purifying human PMNs and quantifying S. aureus cytotoxicity against them.
- To provide methods for assessing S. aureus leukocidin activity on PMN plasma membranes.
Main Methods:
- Isolation of human PMNs and serum via density centrifugation.
- Cytotoxicity assays using extracellular S. aureus proteins and live S. aureus.
- Flow cytometry analysis with propidium iodide to measure membrane integrity disruption.
Main Results:
- Established procedures for human PMN purification and S. aureus cytotoxicity assessment.
- Quantified S. aureus leukocidin-mediated disruption of PMN plasma membranes.
- Demonstrated the utility of propidium iodide staining for measuring cell death.
Conclusions:
- The described methods enable rapid evaluation of S. aureus cytotoxicity against primary human PMNs.
- These protocols can be adapted to study various host-pathogen interactions.
- The findings contribute to understanding S. aureus pathogenesis and immune evasion strategies.
