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

Nocodazole and chloramphenicol inhibit megasome formation and increase intracellular killing of Hp. Peritoneal macrophages and Hp were treated with 2 μg/ml nocodazole, 30–100 μg/ml chloramphenicol, or 100 μg/ml cycloheximide as described in Materials and Methods. Megasome formation and megasome size was scored using LM and TEM. Phagocytic killing was assayed as described above. (A) Nocodazole and chloramphenicol inhibit megasome formation. Macrophages and Hp were treated with nocodazole (Nocod.), chloramphenicol (Clr.), or cycloheximide (Chx.) as indicated, and samples were fixed-processed for LM and TEM 2 h after initiation of phagocytosis. The graphs show the number of megasomes as a percentage of all Hp phagosomes. Top panel: megasomes were scored in fixed and permeabilized cells using LM. Data shown are the average ± SD of three to six independent experiments performed in triplicate. Bottom panel, effect of 100 μg/ml chloramphenicol and 2 μg/ml nocodazole on megasome formation as judged by TEM. Con., control. (B) Effect of 2 μg/ml nocodazole and 100 μg/ml chloramphenicol on megasome size. Macrophages were fixed and processed for TEM 2 h after initiation of phagocytosis. “Frequency” indicates the number of megasomes containing 2 Hp (black bars), 3–5 Hp (white bars), or >5 Hp (hatched bars). The size of 2 h megasomes in control macrophages is shown in  Fig. 5. (C) Macrophages and Hp 11637 were left untreated or incubated with 2 μg/ml nocodazole or 100 μg/ml chloramphenicol, and phagocytic killing was measured after 0.5–20 h. Data shown are the average ± SD of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195807&req=5

Figure 7: Nocodazole and chloramphenicol inhibit megasome formation and increase intracellular killing of Hp. Peritoneal macrophages and Hp were treated with 2 μg/ml nocodazole, 30–100 μg/ml chloramphenicol, or 100 μg/ml cycloheximide as described in Materials and Methods. Megasome formation and megasome size was scored using LM and TEM. Phagocytic killing was assayed as described above. (A) Nocodazole and chloramphenicol inhibit megasome formation. Macrophages and Hp were treated with nocodazole (Nocod.), chloramphenicol (Clr.), or cycloheximide (Chx.) as indicated, and samples were fixed-processed for LM and TEM 2 h after initiation of phagocytosis. The graphs show the number of megasomes as a percentage of all Hp phagosomes. Top panel: megasomes were scored in fixed and permeabilized cells using LM. Data shown are the average ± SD of three to six independent experiments performed in triplicate. Bottom panel, effect of 100 μg/ml chloramphenicol and 2 μg/ml nocodazole on megasome formation as judged by TEM. Con., control. (B) Effect of 2 μg/ml nocodazole and 100 μg/ml chloramphenicol on megasome size. Macrophages were fixed and processed for TEM 2 h after initiation of phagocytosis. “Frequency” indicates the number of megasomes containing 2 Hp (black bars), 3–5 Hp (white bars), or >5 Hp (hatched bars). The size of 2 h megasomes in control macrophages is shown in Fig. 5. (C) Macrophages and Hp 11637 were left untreated or incubated with 2 μg/ml nocodazole or 100 μg/ml chloramphenicol, and phagocytic killing was measured after 0.5–20 h. Data shown are the average ± SD of four independent experiments.

Mentions: Intracellular organelle transport is mediated by the cytoskeleton, and previous studies have shown that phagosomes move bidirectionally along microtubular tracts in macrophages 35 36. As clustering and fusion of Hp phagosomes appeared to be highly dynamic, we explored the role of MTs in this process. MTs were not essential for Hp phagocytosis, and the rate of Hp ingestion was not altered in nocodazole-treated cells ( Fig. 6). In contrast, we found that depolymerization of macrophage MTs with either nocodazole or colcemid inhibited megasome formation by ∼90% ( Fig. 7 A), and the few megasomes that did form were reduced in size (compare Fig. 7 B to Fig. 5, Fig. 2 h time point). Moreover, in the absence of intact MTs, 99% of ingested Hp were killed ( Fig. 7 C). However, neither drug impaired Hp viability in the absence of macrophages (data not shown). Efficient phagocytic killing of Hp in the absence of MTs was somewhat surprising, as MT-destabilizing drugs can reduce killing of some microorganisms via their ability to block phagosome–lysosome fusion 37. Nevertheless, we found that Ye was also killed by nocodazole-treated macrophages (2 logs killing at 3 h in the presence of nocodazole vs. 1.5 logs killing in the absence of the drug, n = 3). These data suggest that nonlysosomal killing mechanisms were bacteriocidal under these conditions. Although the exact nature of the Hp phagosome remains undefined, our data indicate that megasome formation is important for Hp 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)

Nocodazole and chloramphenicol inhibit megasome formation and increase intracellular killing of Hp. Peritoneal macrophages and Hp were treated with 2 μg/ml nocodazole, 30–100 μg/ml chloramphenicol, or 100 μg/ml cycloheximide as described in Materials and Methods. Megasome formation and megasome size was scored using LM and TEM. Phagocytic killing was assayed as described above. (A) Nocodazole and chloramphenicol inhibit megasome formation. Macrophages and Hp were treated with nocodazole (Nocod.), chloramphenicol (Clr.), or cycloheximide (Chx.) as indicated, and samples were fixed-processed for LM and TEM 2 h after initiation of phagocytosis. The graphs show the number of megasomes as a percentage of all Hp phagosomes. Top panel: megasomes were scored in fixed and permeabilized cells using LM. Data shown are the average ± SD of three to six independent experiments performed in triplicate. Bottom panel, effect of 100 μg/ml chloramphenicol and 2 μg/ml nocodazole on megasome formation as judged by TEM. Con., control. (B) Effect of 2 μg/ml nocodazole and 100 μg/ml chloramphenicol on megasome size. Macrophages were fixed and processed for TEM 2 h after initiation of phagocytosis. “Frequency” indicates the number of megasomes containing 2 Hp (black bars), 3–5 Hp (white bars), or >5 Hp (hatched bars). The size of 2 h megasomes in control macrophages is shown in  Fig. 5. (C) Macrophages and Hp 11637 were left untreated or incubated with 2 μg/ml nocodazole or 100 μg/ml chloramphenicol, and phagocytic killing was measured after 0.5–20 h. Data shown are the average ± SD of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Nocodazole and chloramphenicol inhibit megasome formation and increase intracellular killing of Hp. Peritoneal macrophages and Hp were treated with 2 μg/ml nocodazole, 30–100 μg/ml chloramphenicol, or 100 μg/ml cycloheximide as described in Materials and Methods. Megasome formation and megasome size was scored using LM and TEM. Phagocytic killing was assayed as described above. (A) Nocodazole and chloramphenicol inhibit megasome formation. Macrophages and Hp were treated with nocodazole (Nocod.), chloramphenicol (Clr.), or cycloheximide (Chx.) as indicated, and samples were fixed-processed for LM and TEM 2 h after initiation of phagocytosis. The graphs show the number of megasomes as a percentage of all Hp phagosomes. Top panel: megasomes were scored in fixed and permeabilized cells using LM. Data shown are the average ± SD of three to six independent experiments performed in triplicate. Bottom panel, effect of 100 μg/ml chloramphenicol and 2 μg/ml nocodazole on megasome formation as judged by TEM. Con., control. (B) Effect of 2 μg/ml nocodazole and 100 μg/ml chloramphenicol on megasome size. Macrophages were fixed and processed for TEM 2 h after initiation of phagocytosis. “Frequency” indicates the number of megasomes containing 2 Hp (black bars), 3–5 Hp (white bars), or >5 Hp (hatched bars). The size of 2 h megasomes in control macrophages is shown in Fig. 5. (C) Macrophages and Hp 11637 were left untreated or incubated with 2 μg/ml nocodazole or 100 μg/ml chloramphenicol, and phagocytic killing was measured after 0.5–20 h. Data shown are the average ± SD of four independent experiments.
Mentions: Intracellular organelle transport is mediated by the cytoskeleton, and previous studies have shown that phagosomes move bidirectionally along microtubular tracts in macrophages 35 36. As clustering and fusion of Hp phagosomes appeared to be highly dynamic, we explored the role of MTs in this process. MTs were not essential for Hp phagocytosis, and the rate of Hp ingestion was not altered in nocodazole-treated cells ( Fig. 6). In contrast, we found that depolymerization of macrophage MTs with either nocodazole or colcemid inhibited megasome formation by ∼90% ( Fig. 7 A), and the few megasomes that did form were reduced in size (compare Fig. 7 B to Fig. 5, Fig. 2 h time point). Moreover, in the absence of intact MTs, 99% of ingested Hp were killed ( Fig. 7 C). However, neither drug impaired Hp viability in the absence of macrophages (data not shown). Efficient phagocytic killing of Hp in the absence of MTs was somewhat surprising, as MT-destabilizing drugs can reduce killing of some microorganisms via their ability to block phagosome–lysosome fusion 37. Nevertheless, we found that Ye was also killed by nocodazole-treated macrophages (2 logs killing at 3 h in the presence of nocodazole vs. 1.5 logs killing in the absence of the drug, n = 3). These data suggest that nonlysosomal killing mechanisms were bacteriocidal under these conditions. Although the exact nature of the Hp phagosome remains undefined, our data indicate that megasome formation is important for Hp 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