Limits...
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.

Show MeSH

Related in: MedlinePlus

Ingestion of Hp by macrophages is delayed relative to bacterial binding. Phagocytosis of Hp 11637 or Ye by peritoneal macrophages was synchronized using centrifugation. After incubation at 37°C for the indicated times, forming phagosomes were detected by staining fixed and permeabilized macrophages with FITC– or rhodamine–phalloidin, and cell-associated organisms were detected using phase contrast microscopy. (A) Representative forming phagosomes containing Hp or Ye. Left column, phase contrast; Right column, F-actin. Forming phagosomes containing Ye were abundant after 0.5 min at 37°C (arrows, bottom panels). Actin rearrangements were not detected beneath bound Hp after 2 min at 37°C (arrows, top panels); however, numerous Hp phagosomes were detected after 4 min at 37°C (arrows, center panels). (B) Kinetics of phagosome formation and bacterial ingestion. Adherent macrophages ingested Hp or Ye for 0–15 min at 37°C before processing for IFM. F-actin was detected as in A, and the results are expressed as the percentage of actin-positive cell-associated bacteria over time. The total number of cell-associated bacteria per 100 macrophages did not change significantly over the time course of the experiment: 800 ± 87 Hp and 771 ± 106 Ye at 1 min, and 882 ± 91 Hp and 800 ± 92 Ye at 15 min. Data shown are the average ± SD from three independent experiments conducted in triplicate. At least 300 bacteria were scored per sample per time. Comparable data were obtained using J774 cells or Hp 60190 (not shown).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195807&req=5

Figure 2: Ingestion of Hp by macrophages is delayed relative to bacterial binding. Phagocytosis of Hp 11637 or Ye by peritoneal macrophages was synchronized using centrifugation. After incubation at 37°C for the indicated times, forming phagosomes were detected by staining fixed and permeabilized macrophages with FITC– or rhodamine–phalloidin, and cell-associated organisms were detected using phase contrast microscopy. (A) Representative forming phagosomes containing Hp or Ye. Left column, phase contrast; Right column, F-actin. Forming phagosomes containing Ye were abundant after 0.5 min at 37°C (arrows, bottom panels). Actin rearrangements were not detected beneath bound Hp after 2 min at 37°C (arrows, top panels); however, numerous Hp phagosomes were detected after 4 min at 37°C (arrows, center panels). (B) Kinetics of phagosome formation and bacterial ingestion. Adherent macrophages ingested Hp or Ye for 0–15 min at 37°C before processing for IFM. F-actin was detected as in A, and the results are expressed as the percentage of actin-positive cell-associated bacteria over time. The total number of cell-associated bacteria per 100 macrophages did not change significantly over the time course of the experiment: 800 ± 87 Hp and 771 ± 106 Ye at 1 min, and 882 ± 91 Hp and 800 ± 92 Ye at 15 min. Data shown are the average ± SD from three independent experiments conducted in triplicate. At least 300 bacteria were scored per sample per time. Comparable data were obtained using J774 cells or Hp 60190 (not shown).

Mentions: Phagosome morphology and the kinetics of bacterial ingestion were followed using FITC– or rhodamine–phalloidin and fluorescence microscopy ( Fig. 2). As neither Ye nor Hp bound to macrophages at 4°C, phagocytosis was synchronized using centrifugation. Ye, which binds to β1 integrins 34, was rapidly internalized into close-fitting conventional phagosomes ( Fig. 2). The kinetics of Ye uptake were similar to those we obtained previously for zymosan, IgG beads, and complement-opsonized particles 24 25. Hp 11637 and 60190 were also detected in close-fitting phagosomes. However, unlike with Ye or other particles, both actin rearrangements beneath attached Hp and phagosome formation were significantly delayed relative to bacterial binding ( Fig. 2). Thus, most Ye were phagocytosed within 0.5–1 min, whereas actin polymerization in the vicinity of bound Hp was negligible until 3–4 min and peaked at 5–7 min. Ingestion kinetics similar to those shown in Fig. 2 (i.e., a delay with virulent Hp) were also obtained using Abs to the cytoskeletal protein talin or using the double-immunofluorescence method (data not shown). Moreover, in contrast to viable organisms, ingestion of dead Hp resembled Ye and occurred without a lag; 81.3 ± 7.0% of cell-associated heat-killed Hp were in actin-positive phagosomes after 1 min at 37°C (n = 3). Neither the receptor that mediates phagocytosis of Hp nor the accompanying signaling events have been elucidated. Nevertheless, it is tempting to speculate that delayed uptake of Hp may reflect alterations of the internalization process that are important for bacterial survival in macrophages.


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)

Ingestion of Hp by macrophages is delayed relative to bacterial binding. Phagocytosis of Hp 11637 or Ye by peritoneal macrophages was synchronized using centrifugation. After incubation at 37°C for the indicated times, forming phagosomes were detected by staining fixed and permeabilized macrophages with FITC– or rhodamine–phalloidin, and cell-associated organisms were detected using phase contrast microscopy. (A) Representative forming phagosomes containing Hp or Ye. Left column, phase contrast; Right column, F-actin. Forming phagosomes containing Ye were abundant after 0.5 min at 37°C (arrows, bottom panels). Actin rearrangements were not detected beneath bound Hp after 2 min at 37°C (arrows, top panels); however, numerous Hp phagosomes were detected after 4 min at 37°C (arrows, center panels). (B) Kinetics of phagosome formation and bacterial ingestion. Adherent macrophages ingested Hp or Ye for 0–15 min at 37°C before processing for IFM. F-actin was detected as in A, and the results are expressed as the percentage of actin-positive cell-associated bacteria over time. The total number of cell-associated bacteria per 100 macrophages did not change significantly over the time course of the experiment: 800 ± 87 Hp and 771 ± 106 Ye at 1 min, and 882 ± 91 Hp and 800 ± 92 Ye at 15 min. Data shown are the average ± SD from three independent experiments conducted in triplicate. At least 300 bacteria were scored per sample per time. Comparable data were obtained using J774 cells or Hp 60190 (not shown).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Ingestion of Hp by macrophages is delayed relative to bacterial binding. Phagocytosis of Hp 11637 or Ye by peritoneal macrophages was synchronized using centrifugation. After incubation at 37°C for the indicated times, forming phagosomes were detected by staining fixed and permeabilized macrophages with FITC– or rhodamine–phalloidin, and cell-associated organisms were detected using phase contrast microscopy. (A) Representative forming phagosomes containing Hp or Ye. Left column, phase contrast; Right column, F-actin. Forming phagosomes containing Ye were abundant after 0.5 min at 37°C (arrows, bottom panels). Actin rearrangements were not detected beneath bound Hp after 2 min at 37°C (arrows, top panels); however, numerous Hp phagosomes were detected after 4 min at 37°C (arrows, center panels). (B) Kinetics of phagosome formation and bacterial ingestion. Adherent macrophages ingested Hp or Ye for 0–15 min at 37°C before processing for IFM. F-actin was detected as in A, and the results are expressed as the percentage of actin-positive cell-associated bacteria over time. The total number of cell-associated bacteria per 100 macrophages did not change significantly over the time course of the experiment: 800 ± 87 Hp and 771 ± 106 Ye at 1 min, and 882 ± 91 Hp and 800 ± 92 Ye at 15 min. Data shown are the average ± SD from three independent experiments conducted in triplicate. At least 300 bacteria were scored per sample per time. Comparable data were obtained using J774 cells or Hp 60190 (not shown).
Mentions: Phagosome morphology and the kinetics of bacterial ingestion were followed using FITC– or rhodamine–phalloidin and fluorescence microscopy ( Fig. 2). As neither Ye nor Hp bound to macrophages at 4°C, phagocytosis was synchronized using centrifugation. Ye, which binds to β1 integrins 34, was rapidly internalized into close-fitting conventional phagosomes ( Fig. 2). The kinetics of Ye uptake were similar to those we obtained previously for zymosan, IgG beads, and complement-opsonized particles 24 25. Hp 11637 and 60190 were also detected in close-fitting phagosomes. However, unlike with Ye or other particles, both actin rearrangements beneath attached Hp and phagosome formation were significantly delayed relative to bacterial binding ( Fig. 2). Thus, most Ye were phagocytosed within 0.5–1 min, whereas actin polymerization in the vicinity of bound Hp was negligible until 3–4 min and peaked at 5–7 min. Ingestion kinetics similar to those shown in Fig. 2 (i.e., a delay with virulent Hp) were also obtained using Abs to the cytoskeletal protein talin or using the double-immunofluorescence method (data not shown). Moreover, in contrast to viable organisms, ingestion of dead Hp resembled Ye and occurred without a lag; 81.3 ± 7.0% of cell-associated heat-killed Hp were in actin-positive phagosomes after 1 min at 37°C (n = 3). Neither the receptor that mediates phagocytosis of Hp nor the accompanying signaling events have been elucidated. Nevertheless, it is tempting to speculate that delayed uptake of Hp may reflect alterations of the internalization process that are important for bacterial survival in macrophages.

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