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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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Metabolic activity is altered in H. pylori cultured with 1% DFMO.Cultures were grown for 24 h and bacterial samples were removed at the indicated time points. Relative ATP levels were measured in bacterial lysates using CellTiter-Glo, a luciferase-based assay kit. (A) Luminescence (Lum) units, proportional to ATP concentration, were calculated for equivolumetric samples. (B) Luminescence units per 106 bacteria at each time point were calculated using the data from panel A, the observed OD600 of the culture, and the equation derived from Figure 5D. Each data point is the average of 3 experiments with the standard errors shown. For both graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.
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pone-0017510-g006: Metabolic activity is altered in H. pylori cultured with 1% DFMO.Cultures were grown for 24 h and bacterial samples were removed at the indicated time points. Relative ATP levels were measured in bacterial lysates using CellTiter-Glo, a luciferase-based assay kit. (A) Luminescence (Lum) units, proportional to ATP concentration, were calculated for equivolumetric samples. (B) Luminescence units per 106 bacteria at each time point were calculated using the data from panel A, the observed OD600 of the culture, and the equation derived from Figure 5D. Each data point is the average of 3 experiments with the standard errors shown. For both graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.

Mentions: Monitoring bacterial growth by optical density has inherent limitations, as both live and dead cells can remain in suspension and contribute to an apparent increase in the number of growing bacteria. We attempted to circumvent this problem by quantifying viable cells by diluting and plating the culture on solid medium. Although we did begin to see a decrease in the number of culturable bacteria after 8 h of culture (Figure 1C), optical density levels diverged 4 h prior (Figure 1B) and significant die-off of H. pylori was not seen until 24 h of culture with 1% DFMO. Our inference was that rather than having a bactericidal effect, DFMO was altering cell metabolism, which led to a significant decrease in growth rate. To determine if this was the case, we utilized a luciferase-based assay to measure ATP levels in bacterial lysates. Unexpectedly, rather than inducing a decrease in ATP, DFMO had the opposite effect. When we analyzed equal volumes of cultures grown with or without 1% DFMO, we observed a significant increase in ATP levels beginning only 1 h after initiation (Figure 6A). ATP levels per microliter increased steadily over time for both cultures until the 24 h time point, when levels in DFMO-containing cultures leveled off. For control bacteria there was obvious parallelism between the ATP levels and the OD600 growth curve shown in Figure 1B, but this was not seen for bacteria exposed to DFMO. As ATP levels can be used as a surrogate measure of cell numbers—for comparably grown cultures—we presumed that the observed increase simply indicated continued growth of H. pylori. To eliminate this uncertainty we used the optical density of the cultures, and the equation derived from Figure 5D, to calculate the ATP-dependent luminescence per 106 bacteria. This transformation demonstrated that the effect of DFMO was even greater than initially perceived. When compared to control cultures, DFMO induced a 1.5-fold increase in luminescence by 1 h that grew to 3.4-fold by 12 h (p<0.001; Figure 6B). We also observed that while the control culture maintained a relatively steady level of ATP/106 bacteria throughout the entire time course (405.8±28.8 luminescence units), large dynamic changes were seen in the 1% DFMO cultures, suggesting that the chemical drastically altered the metabolism of H. pylori.


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)

Metabolic activity is altered in H. pylori cultured with 1% DFMO.Cultures were grown for 24 h and bacterial samples were removed at the indicated time points. Relative ATP levels were measured in bacterial lysates using CellTiter-Glo, a luciferase-based assay kit. (A) Luminescence (Lum) units, proportional to ATP concentration, were calculated for equivolumetric samples. (B) Luminescence units per 106 bacteria at each time point were calculated using the data from panel A, the observed OD600 of the culture, and the equation derived from Figure 5D. Each data point is the average of 3 experiments with the standard errors shown. For both graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046249&req=5

pone-0017510-g006: Metabolic activity is altered in H. pylori cultured with 1% DFMO.Cultures were grown for 24 h and bacterial samples were removed at the indicated time points. Relative ATP levels were measured in bacterial lysates using CellTiter-Glo, a luciferase-based assay kit. (A) Luminescence (Lum) units, proportional to ATP concentration, were calculated for equivolumetric samples. (B) Luminescence units per 106 bacteria at each time point were calculated using the data from panel A, the observed OD600 of the culture, and the equation derived from Figure 5D. Each data point is the average of 3 experiments with the standard errors shown. For both graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.
Mentions: Monitoring bacterial growth by optical density has inherent limitations, as both live and dead cells can remain in suspension and contribute to an apparent increase in the number of growing bacteria. We attempted to circumvent this problem by quantifying viable cells by diluting and plating the culture on solid medium. Although we did begin to see a decrease in the number of culturable bacteria after 8 h of culture (Figure 1C), optical density levels diverged 4 h prior (Figure 1B) and significant die-off of H. pylori was not seen until 24 h of culture with 1% DFMO. Our inference was that rather than having a bactericidal effect, DFMO was altering cell metabolism, which led to a significant decrease in growth rate. To determine if this was the case, we utilized a luciferase-based assay to measure ATP levels in bacterial lysates. Unexpectedly, rather than inducing a decrease in ATP, DFMO had the opposite effect. When we analyzed equal volumes of cultures grown with or without 1% DFMO, we observed a significant increase in ATP levels beginning only 1 h after initiation (Figure 6A). ATP levels per microliter increased steadily over time for both cultures until the 24 h time point, when levels in DFMO-containing cultures leveled off. For control bacteria there was obvious parallelism between the ATP levels and the OD600 growth curve shown in Figure 1B, but this was not seen for bacteria exposed to DFMO. As ATP levels can be used as a surrogate measure of cell numbers—for comparably grown cultures—we presumed that the observed increase simply indicated continued growth of H. pylori. To eliminate this uncertainty we used the optical density of the cultures, and the equation derived from Figure 5D, to calculate the ATP-dependent luminescence per 106 bacteria. This transformation demonstrated that the effect of DFMO was even greater than initially perceived. When compared to control cultures, DFMO induced a 1.5-fold increase in luminescence by 1 h that grew to 3.4-fold by 12 h (p<0.001; Figure 6B). We also observed that while the control culture maintained a relatively steady level of ATP/106 bacteria throughout the entire time course (405.8±28.8 luminescence units), large dynamic changes were seen in the 1% DFMO cultures, suggesting that the chemical drastically altered the metabolism of H. pylori.

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