Anti-Listeria monocytogenes Activity of Human Neutrophils

Abstract

Listeria monocytogenes is a facultative intracellular pathogen, which invades and replicates within a variety of cells, causing a severe foodborne illness in humans with a high fatality rate of 30%. The major virulence factor of L. monocytogenes is listeriolysin O (LLO), a secreted pore-forming toxin that perforates the phagosomal membrane and mediates bacterial escape to the cytoplasm, where replication occurs. Neutrophils and macrophages are professional phagocytes that are essential to the innate immune defense. They ingest microorganisms into phagosomes, which collect antimicrobial molecules to rapidly and efficiently kill microorganisms. It is well known that L. monocytogenes escapes from the phagosome in macrophages, avoiding their killing mechanisms and allowing the bacteria to multiply in the cytoplasm. However, little is known about the interactions of L. monocytogenes with neutrophils. The aim of my project was to determine whether neutrophils, isolated from human peripheral blood, are able to ingest and kill L. monocytogenes, or whether LLO can defeat the neutrophil’s antimicrobial mechanisms. This was accomplished by monitoring the efficiencies of wild type and LLO-deficient strains of L. monocytogenes phagocytosis and killing by neutrophils in comparison to that of macrophages (cell line RAW 264.7). Fluorescence-based microscopy methods were used to monitor phagocytic uptake and intracellular proliferation of bacteria. Intracellular and total (extracellular and intracellular) killing was measured using a gentamicin survival assay. Future directions include analysis of bacterial escape from the phagosome and/or fusion of neutrophil granules with the phagosome using immunofluorescence microscopy.