SummaryIt has long been known from the results of ultrastructural studies that complement-and inmmnoglobulin G (IgG)-opsonized particles are phagocytosed differently by macrophages (Kaplan, G. 1977. &and. J. bnmunol. 6:797-807). Complement-opsonized particles sink into the cell, whereas IgG-coated particles are engulfed by lamellipodia, which project from the cell surface. The molecular basis for these differences is unknown. We used indirect inmmnofluorescence and confocal microscopy to examine how cytoskeletal proteins associate with phagosomes containing complement-opsonized zymosan (COZ) particles or IgG beads in phorbol-myristateacetate-treated peritoneal macrophages. During ingestion of COZ, punctate structures rich in F-actin, vinculin, a-actinin, paxillin, and phosphotyrosine-containing proteins are distributed over the phagosome surface. These loci are detected beneath bound COZ within 30 s of warnfing the cells to 37~ and their formation requires active protein kinase C. By contrast, during Fc receptor-mediated phagocytosis, all proteins exanfined were uniformly distributed on or near the phagosome surface. Moreover, ingestion of IgG beads was blocked by tyrosine kinase inhibitors, whereas phagocytosis of COZ was not. Thus, the signals required for particle ingestion, and the arrangement of cytoskeletal proteins on the phagosome surface, vary depending upon which phagocytic receptor is engaged. Moreover, complement receptor (CR)-mediated internalization required intact microtubules and was accompanied by the accumulation of vesicles beneath the forming phagosome, suggesting that membrane trafficking plays a key role in C1K-mediated phagocytosis.

Francisella tularensis is a Gram-negative bacterium and the causative agent of tularemia. Recent data indicate that F. tularensis replicates inside macrophages, but its fate in other cell types, including human neutrophils, is unclear. We now show that F. tularensis live vaccine strain (LVS), opsonized with normal human serum, was rapidly ingested by neutrophils but was not eliminated. Moreover, evasion of intracellular killing can be explained, in part, by disruption of the respiratory burst. As judged by luminol-enhanced chemiluminescence and nitroblue tetrazolium staining, neutrophils infected with live F. tularensis did not generate reactive oxygen species. Confocal microscopy demonstrated that NADPH oxidase assembly was disrupted, and LVS phagosomes did not acquire gp91/p22 phox or p47/p67 phox . At the same time, F. tularensis also impaired neutrophil activation by heterologous stimuli such as phorbol esters and opsonized zymosan particles. Later in infection, LVS escaped the phagosome, and live organisms persisted in the neutrophil cytosol for at least 12 h. To our knowledge, our data are the first demonstration of a facultative intracellular pathogen, which disrupts the oxidative burst and escapes the phagosome to evade elimination inside neutrophils, and as such, our data define a novel mechanism of virulence.

Tularemia is a zoonosis of humans caused by infection with the facultative intracellular bacterium Francisella tularensis. Interest in F. tularensis has increased markedly in the past few years because of its potential use as an agent of bioterrorism. Five subspecies of this organism are found in the Northern hemisphere, but only F. tularensis subsp. tularensis and subsp. holarctica cause disease in humans. This review summarizes what is known about the pathogenesis of tularemia with a focus on bacterial surface components such as lipopolysaccharide and capsule as well as information obtained from the F. tularensis subsp. tularensis SCHU S4 genome. In particular, the mechanisms of action of recently identified virulence factors are discussed in the context of bacterial replication in macrophages and manipulation of the host inflammatory response. Throughout this report shared and unique features of F. tularensis subsp. tularensis, subsp. holarctica, and subsp. novicida are discussed.

Helicobacter pylori colonizes the gastric epithelium of ∼50% of the world's population and plays a causative role in the development of gastric and duodenal ulcers. H. pylori is phagocytosed by mononuclear phagocytes, but the internalized bacteria are not killed and the reasons for this host defense defect are unclear. We now show using immunofluorescence and electron microscopy that H. pylori employs an unusual mechanism to avoid phagocytic killing: delayed entry followed by homotypic phagosome fusion. Unopsonized type I H. pylori bound readily to macrophages and were internalized into actin-rich phagosomes after a lag of ∼4 min. Although early (10 min) phagosomes contained single bacilli, H. pylori phagosomes coalesced over the next ∼2 h. The resulting “megasomes” contained multiple viable organisms and were stable for 24 h. Phagosome–phagosome fusion required bacterial protein synthesis and intact host microtubules, and both chloramphenicol and nocodazole increased killing of intracellular H. pylori. Type II strains of H. pylori are less virulent and lack the cag pathogenicity island. In contrast to type I strains, type II H. pylori were rapidly ingested and killed by macrophages and did not stimulate megasome formation. Collectively, our data suggest that megasome formation is an important feature of H. pylori pathogenesis.