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Updated: Mar 3, 2026
Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms
Published on: June 8, 2022
Lysis of human neutrophils by community-associated methicillin-resistant Staphylococcus aureus
Mallary C Greenlee-Wacker1, Silvie Kremserová2,3,4, William M Nauseef2,3,4
1Department of Biology, Central Michigan University, Mount Pleasant, MI.
Abstract:
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes infections associated with extensive tissue damage and necrosis. In vitro, human neutrophils fed CA-MRSA lyse by an unknown mechanism that is inhibited by necrostatin-1, an allosteric inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK-1). RIPK-1 figures prominently in necroptosis, a specific form of programmed cell death dependent on RIPK-1, RIPK-3, and the mixed-lineage kinase-like protein (MLKL). We previously reported that necrostatin-1 inhibits lysis of human neutrophils fed CA-MRSA and attributed the process to necroptosis. We now extend our studies to examine additional components in the programmed cell death pathway to test the hypothesis that neutrophils fed CA-MRSA undergo necroptosis. Lysis of neutrophils fed CA-MRSA was independent of tumor necrosis factor α, active RIPK-1, and MLKL, but dependent on active RIPK-3. Human neutrophils fed CA-MRSA lacked phosphorylated RIPK-1, as well as phosphorylated or oligomerized MLKL. Neutrophils fed CA-MRSA possessed cytoplasmic complexes that included inactive caspase 8, RIPK-1, and RIPK-3, and the composition of the complex remained stable over time. Together, these data suggest that neutrophils fed CA-MRSA underwent a novel form of lytic programmed cell death via a mechanism that required RIPK-3 activity, but not active RIPK-1 or MLKL, and therefore was distinct from necroptosis. Targeting the molecular pathways that culminate in lysis of neutrophils during CA-MRSA infection may serve as a novel therapeutic intervention to limit the associated tissue damage.
Insights
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes severe infections. Neutrophil lysis during CA-MRSA infection involves RIPK-3 but not RIPK-1 or MLKL, indicating a novel cell death pathway distinct from necroptosis.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections lead to significant tissue damage.
- Neutrophil lysis, previously attributed to necroptosis, is observed in vitro when neutrophils are exposed to CA-MRSA.
- Necrostatin-1, a RIPK-1 inhibitor, was found to inhibit this lysis, suggesting a role for programmed cell death.
Purpose of the Study:
- To investigate the molecular mechanisms underlying neutrophil lysis induced by CA-MRSA.
- To determine if the observed neutrophil lysis is indeed necroptosis by examining key components of the programmed cell death pathway.
- To identify novel therapeutic targets for mitigating tissue damage in CA-MRSA infections.
Main Methods:
- Human neutrophils were incubated with CA-MRSA in vitro.
- Analysis of programmed cell death markers including receptor-interacting serine/threonine kinase 1 (RIPK-1), RIPK-3, mixed-lineage kinase-like protein (MLKL), and caspase 8.
- Assessment of protein phosphorylation and complex formation within neutrophils.
Main Results:
- Neutrophil lysis upon CA-MRSA exposure was independent of tumor necrosis factor α, active RIPK-1, and MLKL.
- Lysis was dependent on active RIPK-3.
- Phosphorylated RIPK-1 and MLKL were absent, and neutrophils contained cytoplasmic complexes with inactive caspase 8, RIPK-1, and RIPK-3.
Conclusions:
- Neutrophil lysis during CA-MRSA infection is a novel form of programmed cell death.
- This pathway requires RIPK-3 activity but is distinct from classical necroptosis as it does not involve active RIPK-1 or MLKL.
- Targeting this RIPK-3-dependent pathway could offer a new therapeutic strategy against CA-MRSA-induced tissue damage.
