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Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction.

Barry DP, Asim M, Leiman DA, de Sablet T, Singh K, Casero RA, Chaturvedi R, Wilson KT - PLoS ONE (2011)

Bottom Line: We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner.H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism.These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

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DFMO suppresses H. pylori CagA expression and its translocation into gastric epithelial cells.H. pylori strain 60190 was grown for 12 h with or without DFMO supplementation. (A) Western blot analysis of CagA and UreB expression in bacteria. An isogenic 60190 cagA mutant was used as a control. (B) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 6 h with these bacteria or uninfected as a control. (C) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 12 h or controls.
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pone-0017510-g008: DFMO suppresses H. pylori CagA expression and its translocation into gastric epithelial cells.H. pylori strain 60190 was grown for 12 h with or without DFMO supplementation. (A) Western blot analysis of CagA and UreB expression in bacteria. An isogenic 60190 cagA mutant was used as a control. (B) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 6 h with these bacteria or uninfected as a control. (C) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 12 h or controls.

Mentions: H. pylori possesses a number of virulence factors known to be important for the induction of disease during infection. One of the best described is the effector molecule CagA, which has been implicated in the disruption of tight junctions and alteration of cellular morphology [46]. As infection with CagA-expressing strains of H. pylori is associated with worse clinical outcome in patients, namely higher rates of peptic ulcer disease and gastric cancer [46], [47], [48], [49], we sought to determine if DFMO treatment could affect the production and function of this bacterial protein. We utilized H. pylori 60190 in this set of experiments, since it has been reported that SS1 has an incomplete cag pathogenicity island (cag-PAI) [50]. Bacteria cultured for 12 h in the presence of 1% DFMO exhibited lower levels of CagA protein expression compared to untreated cells (Figure 8A). This effect was not due to a global suppression of protein translation, as levels of UreB, a component of the highly-expressed bacterial urease, were not affected. Although we observed a decrease in CagA protein, mRNA levels were not affected by DFMO (Figure S3A), indicating that it acts in a post-transcriptional manner. This type of control of expression, which can involve altered mRNA stability, has been reported for other H. pylori genes [51], [52], [53]. We measured the stability of cagA from cultures grown with or without DFMO, but did not observe any increase in decay rate; in fact, we found that DFMO improved transcript stability and increased its half-life from 3.4 min to 4.3 min (Figure S3B).


Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction.

Barry DP, Asim M, Leiman DA, de Sablet T, Singh K, Casero RA, Chaturvedi R, Wilson KT - PLoS ONE (2011)

DFMO suppresses H. pylori CagA expression and its translocation into gastric epithelial cells.H. pylori strain 60190 was grown for 12 h with or without DFMO supplementation. (A) Western blot analysis of CagA and UreB expression in bacteria. An isogenic 60190 cagA mutant was used as a control. (B) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 6 h with these bacteria or uninfected as a control. (C) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 12 h or controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046249&req=5

pone-0017510-g008: DFMO suppresses H. pylori CagA expression and its translocation into gastric epithelial cells.H. pylori strain 60190 was grown for 12 h with or without DFMO supplementation. (A) Western blot analysis of CagA and UreB expression in bacteria. An isogenic 60190 cagA mutant was used as a control. (B) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 6 h with these bacteria or uninfected as a control. (C) Western blot analysis of total CagA, tyrosine-phosphorylated CagA, and β-actin in lysates prepared from AGS cells infected for 12 h or controls.
Mentions: H. pylori possesses a number of virulence factors known to be important for the induction of disease during infection. One of the best described is the effector molecule CagA, which has been implicated in the disruption of tight junctions and alteration of cellular morphology [46]. As infection with CagA-expressing strains of H. pylori is associated with worse clinical outcome in patients, namely higher rates of peptic ulcer disease and gastric cancer [46], [47], [48], [49], we sought to determine if DFMO treatment could affect the production and function of this bacterial protein. We utilized H. pylori 60190 in this set of experiments, since it has been reported that SS1 has an incomplete cag pathogenicity island (cag-PAI) [50]. Bacteria cultured for 12 h in the presence of 1% DFMO exhibited lower levels of CagA protein expression compared to untreated cells (Figure 8A). This effect was not due to a global suppression of protein translation, as levels of UreB, a component of the highly-expressed bacterial urease, were not affected. Although we observed a decrease in CagA protein, mRNA levels were not affected by DFMO (Figure S3A), indicating that it acts in a post-transcriptional manner. This type of control of expression, which can involve altered mRNA stability, has been reported for other H. pylori genes [51], [52], [53]. We measured the stability of cagA from cultures grown with or without DFMO, but did not observe any increase in decay rate; in fact, we found that DFMO improved transcript stability and increased its half-life from 3.4 min to 4.3 min (Figure S3B).

Bottom Line: We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner.H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism.These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

Show MeSH
Related in: MedlinePlus