Quantifying the Cytotoxicity of <em>Staphylococcus aureus</em> Against Human Polymorphonuclear Leukocytes

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.

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence

“…A powerful discovery approach has been used to compare the cytolytic activities of secreted S. aureus virulence factors between colonising and bacteraemia isolates, followed by genomics to uncover the bacterial genetic loci linked with high/low cytolytic activity ( Collins et al, 2008 ; Giulieri et al, 2018 ; Laabei et al, 2014 ; Laabei et al, 2021 ; McConville et al, 2022 ). These toxicity analyses use methods that monitor eukaryotic cell viability upon exposure to S. aureus culture supernatants ( Dankoff et al, 2020 ; Das et al, 2016 ; Giulieri et al, 2018 ) and integrate these phenotypes within genome-wide association studies (GWAS) and other comparative bacterial population genomics techniques ( Giulieri et al, 2018 ; Recker et al, 2017 ). However, such toxicity assays are limited in that they focus on exogenous virulence factors that have accumulated in the culture media during bacterial growth.…”

A high-throughput cytotoxicity screening platform revealsagr-independent mutations in bacteraemia-associatedStaphylococcus aureusthat promote intracellular persistence

“…A powerful discovery approach has been used to compare the cytolytic activities of secreted S. aureus virulence factors between colonising and bacteraemia isolates, followed by genomics to uncover the bacterial genetic loci linked with high/low cytolytic activity (Collins et al, 2008; Giulieri et al, 2018; Laabei et al, 2014, 2021; McConville et al, 2022). These toxicity analyses use methods that monitor eukaryotic cell viability upon exposure to S. aureus culture supernatants (Dankoff et al, 2020; Das et al, 2016; Giulieri et al, 2018) and integrate these phenotypes within genome wide association studies (GWAS) and other comparative bacterial population genomics techniques (Giulieri et al, 2018; Recker et al, 2017). However, such toxicity assays are limited in that they focus on exogenous virulence factors that have accumulated in the culture media during bacterial growth.…”