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Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages.

Allen LA, Schlesinger LS, Kang B - J. Exp. Med. (2000)

Bottom Line: The resulting "megasomes" contained multiple viable organisms and were stable for 24 h.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Iowa, Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA. lee-ann-allen@uiowa.edu

ABSTRACT
Helicobacter pylori colonizes the gastric epithelium of approximately 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 approximately 4 min. Although early (10 min) phagosomes contained single bacilli, H. pylori phagosomes coalesced over the next approximately 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.

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Related in: MedlinePlus

Phagocytic killing of Hp by murine macrophages is impaired. Peritoneal macrophages were incubated with Hp 11637 or Ye 8081c at a ratio of 1:25, and phagocytosis was synchronized using centrifugation. As indicated in Materials and Methods, bacterial viability before gentamicin treatment (15 min and 1 h) was determined by vital staining of permeabilized macrophages, whereas viability at later times (2–24 h) was determined by plating macrophage lysates for CFU. Data shown are the average ± SD of three independent experiments. Note that the y-axis is a log scale. Comparable data were obtained using Hp 60190 and J774 cells (not shown). Similar killing curves were generated using 5–100 bacteria per phagocyte (not shown). Mφ, macrophage.
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Figure 1: Phagocytic killing of Hp by murine macrophages is impaired. Peritoneal macrophages were incubated with Hp 11637 or Ye 8081c at a ratio of 1:25, and phagocytosis was synchronized using centrifugation. As indicated in Materials and Methods, bacterial viability before gentamicin treatment (15 min and 1 h) was determined by vital staining of permeabilized macrophages, whereas viability at later times (2–24 h) was determined by plating macrophage lysates for CFU. Data shown are the average ± SD of three independent experiments. Note that the y-axis is a log scale. Comparable data were obtained using Hp 60190 and J774 cells (not shown). Similar killing curves were generated using 5–100 bacteria per phagocyte (not shown). Mφ, macrophage.

Mentions: Previous studies have shown that unopsonized Hp is inefficiently killed by human phagocytes 16 17 18. Similarly, we found that Hp 11637 or 60190 remained viable in resting mouse peritoneal macrophages or J774 cells 24 h after ingestion, whereas avirulent Ye 8081c did not ( Fig. 1). The ability of pathogens such as Mycobacterium tuberculosis 19 21 to disrupt phagosome maturation and phagosome–lysosome fusion is thought to be an important aspect of bacterial virulence and survival. However, the mechanism by which type I strains of Hp resist phagocytic killing is unknown. In this study, we used IFM and TEM to characterize the Hp phagosome.


Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages.

Allen LA, Schlesinger LS, Kang B - J. Exp. Med. (2000)

Phagocytic killing of Hp by murine macrophages is impaired. Peritoneal macrophages were incubated with Hp 11637 or Ye 8081c at a ratio of 1:25, and phagocytosis was synchronized using centrifugation. As indicated in Materials and Methods, bacterial viability before gentamicin treatment (15 min and 1 h) was determined by vital staining of permeabilized macrophages, whereas viability at later times (2–24 h) was determined by plating macrophage lysates for CFU. Data shown are the average ± SD of three independent experiments. Note that the y-axis is a log scale. Comparable data were obtained using Hp 60190 and J774 cells (not shown). Similar killing curves were generated using 5–100 bacteria per phagocyte (not shown). Mφ, macrophage.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195807&req=5

Figure 1: Phagocytic killing of Hp by murine macrophages is impaired. Peritoneal macrophages were incubated with Hp 11637 or Ye 8081c at a ratio of 1:25, and phagocytosis was synchronized using centrifugation. As indicated in Materials and Methods, bacterial viability before gentamicin treatment (15 min and 1 h) was determined by vital staining of permeabilized macrophages, whereas viability at later times (2–24 h) was determined by plating macrophage lysates for CFU. Data shown are the average ± SD of three independent experiments. Note that the y-axis is a log scale. Comparable data were obtained using Hp 60190 and J774 cells (not shown). Similar killing curves were generated using 5–100 bacteria per phagocyte (not shown). Mφ, macrophage.
Mentions: Previous studies have shown that unopsonized Hp is inefficiently killed by human phagocytes 16 17 18. Similarly, we found that Hp 11637 or 60190 remained viable in resting mouse peritoneal macrophages or J774 cells 24 h after ingestion, whereas avirulent Ye 8081c did not ( Fig. 1). The ability of pathogens such as Mycobacterium tuberculosis 19 21 to disrupt phagosome maturation and phagosome–lysosome fusion is thought to be an important aspect of bacterial virulence and survival. However, the mechanism by which type I strains of Hp resist phagocytic killing is unknown. In this study, we used IFM and TEM to characterize the Hp phagosome.

Bottom Line: The resulting "megasomes" contained multiple viable organisms and were stable for 24 h.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Iowa, Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA. lee-ann-allen@uiowa.edu

ABSTRACT
Helicobacter pylori colonizes the gastric epithelium of approximately 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 approximately 4 min. Although early (10 min) phagosomes contained single bacilli, H. pylori phagosomes coalesced over the next approximately 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.

Show MeSH
Related in: MedlinePlus