Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 139

12
Rate and Extent of Helicobacter pylori Phagocytosis
Lee-Ann H. Allen
Summary
Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric epithelium
and plays a causative role in the development of peptic ulcers and gastric cancer. Phago-
cytosis is an element of innate defense used by macrophages and neutrophils to engulf
microorganisms. We and others have shown that strains of H. pylori that contain the cag
pathogenicity island actively retard their entry into phagocytes. Consequently, there is a
lag of several minutes between bacterial binding and the onset of engulfment, and relative
to other particles and microbes, the rate of internalization is slow. Herein, we describe in
detail the use of synchronized phagocytosis and indirect immunofluorescence microscopy
to quantify the rate and extent of H. pylori phagocytosis . This method is appropriate for
primary phagocytes as well as transformed cell lines. More importantly, the effects of
opsonins, virulence factors, and other agents on infection can be measured independent
of bacterial viability or intracellular locale.
Key Words: Macrophage; neutrophil; phagocytosis; immunofluorescence micros-
copy; antibody; opsonin.
1. Introduction
The ability of phagocytes to rapidly ingest and kill large numbers of microor-
ganisms is an essential element of innate defense. Ligation of specific receptors
on macrophages, monocytes and neutrophils activates signaling cascades that
drive local actin polymerization and plasma membrane expansion during phago-
cytosis (1,2) . The morphology of the forming phagosome is dictated by the
receptor engaged and can be modulated further by virulence factors of certain
pathogens (1,2) . Typically, ingestion is very rapid; actin polymerization is