Attachment of Listeria monocytogenes to murine and human phagocytic cells

Listeria monocytogenes is a facultative intracellular pathogen responsible for meningoencephalitis, septicemia, and abortion in susceptible and immunocompromised individuals. Subsequent to colonization and penetration of the gut epithelium, the organism attaches to resident macrophages and replicates intracellularly, thus evading the humoral immune system of the infected host. The mechanism surrounding the precise interactions between L. monocytogenes and macrophages employs bacterial ligands and macrophage receptors which are highly complex and not well understood. The focus of the present study was to investigate the attachment of the organism to murine and human macrophages under opsonin-dependent and opsonin-independent conditions to better understand the pathobiology of this severe pathogen and to potentially develop regimes to more adequately prevent disease. Immunofluorescence (IFA) and enzyme linked immunosorbent assays (ELISA) showed that adhesion of the organism to macrophages regardless of opsonins was inhibited (90%) by N-acetyl neuraminic acid (NAcNeu) and related derivatives. The physical and chemical nature of the cell surface-associated proteins from the pathogen were elucidated by biochemical, electrophoretic, lectin affinity, and soluble binding assays. These studies revealed the presence of several proteins (82, 65, 42, and 33 kDa) involved in facilitating bacterial adhesion to macrophages. Two new bacterial ligands (82, and 65 kDa proteins) were identified and characterized as bacterial glycoproteins. To elucidate the identity of the macrophage receptor(s) involved in listerial attachment, investigations with a phylogenetic and medically similar pathogen, Group B streptococci (GBS) was employed. Competition assays, flow cytometry, and soluble binding experiments revealed that Listeria was able to bind to phorbol myristate acetate-treated and untreated U937 cells in a dose-dependent manner. Complement receptor type 3 was not the primary receptor for attachment of Listeria to polymorphonuclear leukocytes deficient in CR3 expression; previous studies have also shown similar results for GBS. ELISA indicated that Listeria competed effectively with GBS sites on U937 cells and murine macrophages, in a dose-dependent manner. Likewise GBS also prevented the binding of Listeria. Western blot analysis confirmed that Listeria and GBS bind to the same 60 and 55 kDa protein receptors on murine and human macrophages