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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 alters bacterial morphology.Bacterial samples were removed from cultures 6 h after the medium switch depicted in Figure 4B. Gram stains were performed and examined by light microscopy (A) and bacterial pellets were fixed, sectioned, and stained for TEM (B). Note that the labels for panel A also apply to panel B. Scale bars indicate 2 µm in panel A and 1 µm in panel B. (C) Stained bacteria were examined by TEM and cross-sectional areas were measured. For each treatment group the box identifies the median and 25th and 75th percentile values and the whiskers indicate the range of measurements (n = 83–129 measurements per group). ***, p<0.001 between groups indicated. D, a plot of OD600 versus bacterial concentration in CFU/mL was created from the data points of the first 12 h of growth curves depicted in Figures 1B and 1C (n = 9 per treatment). The lines illustrate the best-fit linear regression of each treatment group (control, solid line; 1% DFMO, dashed line) constrained to pass through the origin. The two lines did not significantly differ (p = 0.595). The global best-fit line (not depicted, R2 = 0.80) indicated that an OD600 of 1 was equivalent to 8.11×108 CFU/mL.
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pone-0017510-g005: DFMO alters bacterial morphology.Bacterial samples were removed from cultures 6 h after the medium switch depicted in Figure 4B. Gram stains were performed and examined by light microscopy (A) and bacterial pellets were fixed, sectioned, and stained for TEM (B). Note that the labels for panel A also apply to panel B. Scale bars indicate 2 µm in panel A and 1 µm in panel B. (C) Stained bacteria were examined by TEM and cross-sectional areas were measured. For each treatment group the box identifies the median and 25th and 75th percentile values and the whiskers indicate the range of measurements (n = 83–129 measurements per group). ***, p<0.001 between groups indicated. D, a plot of OD600 versus bacterial concentration in CFU/mL was created from the data points of the first 12 h of growth curves depicted in Figures 1B and 1C (n = 9 per treatment). The lines illustrate the best-fit linear regression of each treatment group (control, solid line; 1% DFMO, dashed line) constrained to pass through the origin. The two lines did not significantly differ (p = 0.595). The global best-fit line (not depicted, R2 = 0.80) indicated that an OD600 of 1 was equivalent to 8.11×108 CFU/mL.

Mentions: Due to the manner of bacterial replication, via binary fission, growth rate can be linked with bacterial size. We therefore determined if DFMO induced any alterations in bacterial morphology. To test this, we sampled bacteria from the 6 h switching experiment and performed Gram stains at 6 h after the medium switch. The bacteria from cultures containing 1% DFMO appeared to be smaller than bacteria from cultures without DFMO (Figure 5A). Due to the low resolution afforded by light microscopy, we also prepared samples of bacteria for transmission electron microscopy. Sections of bacterial pellets were examined (Figure 5B) and the sizes of individual bacteria were determined using NIH ImageJ software. As was suspected from the Gram stains, bacteria cultured in the presence of 1% DFMO were significantly smaller than those cultured in plain broth (p<0.001). We also observed that 6 h after removal of DFMO bacterial size had significantly increased (p<0.001), and that 6 h of culture with DFMO after switching was sufficient to significantly reduce bacterial size (p<0.001) (Figure 5C). These data indicate that H. pylori morphology is rapidly altered by exposure to DFMO, but that the effect disappears once the chemical is removed.


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 alters bacterial morphology.Bacterial samples were removed from cultures 6 h after the medium switch depicted in Figure 4B. Gram stains were performed and examined by light microscopy (A) and bacterial pellets were fixed, sectioned, and stained for TEM (B). Note that the labels for panel A also apply to panel B. Scale bars indicate 2 µm in panel A and 1 µm in panel B. (C) Stained bacteria were examined by TEM and cross-sectional areas were measured. For each treatment group the box identifies the median and 25th and 75th percentile values and the whiskers indicate the range of measurements (n = 83–129 measurements per group). ***, p<0.001 between groups indicated. D, a plot of OD600 versus bacterial concentration in CFU/mL was created from the data points of the first 12 h of growth curves depicted in Figures 1B and 1C (n = 9 per treatment). The lines illustrate the best-fit linear regression of each treatment group (control, solid line; 1% DFMO, dashed line) constrained to pass through the origin. The two lines did not significantly differ (p = 0.595). The global best-fit line (not depicted, R2 = 0.80) indicated that an OD600 of 1 was equivalent to 8.11×108 CFU/mL.
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Related In: Results  -  Collection

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pone-0017510-g005: DFMO alters bacterial morphology.Bacterial samples were removed from cultures 6 h after the medium switch depicted in Figure 4B. Gram stains were performed and examined by light microscopy (A) and bacterial pellets were fixed, sectioned, and stained for TEM (B). Note that the labels for panel A also apply to panel B. Scale bars indicate 2 µm in panel A and 1 µm in panel B. (C) Stained bacteria were examined by TEM and cross-sectional areas were measured. For each treatment group the box identifies the median and 25th and 75th percentile values and the whiskers indicate the range of measurements (n = 83–129 measurements per group). ***, p<0.001 between groups indicated. D, a plot of OD600 versus bacterial concentration in CFU/mL was created from the data points of the first 12 h of growth curves depicted in Figures 1B and 1C (n = 9 per treatment). The lines illustrate the best-fit linear regression of each treatment group (control, solid line; 1% DFMO, dashed line) constrained to pass through the origin. The two lines did not significantly differ (p = 0.595). The global best-fit line (not depicted, R2 = 0.80) indicated that an OD600 of 1 was equivalent to 8.11×108 CFU/mL.
Mentions: Due to the manner of bacterial replication, via binary fission, growth rate can be linked with bacterial size. We therefore determined if DFMO induced any alterations in bacterial morphology. To test this, we sampled bacteria from the 6 h switching experiment and performed Gram stains at 6 h after the medium switch. The bacteria from cultures containing 1% DFMO appeared to be smaller than bacteria from cultures without DFMO (Figure 5A). Due to the low resolution afforded by light microscopy, we also prepared samples of bacteria for transmission electron microscopy. Sections of bacterial pellets were examined (Figure 5B) and the sizes of individual bacteria were determined using NIH ImageJ software. As was suspected from the Gram stains, bacteria cultured in the presence of 1% DFMO were significantly smaller than those cultured in plain broth (p<0.001). We also observed that 6 h after removal of DFMO bacterial size had significantly increased (p<0.001), and that 6 h of culture with DFMO after switching was sufficient to significantly reduce bacterial size (p<0.001) (Figure 5C). These data indicate that H. pylori morphology is rapidly altered by exposure to DFMO, but that the effect disappears once the chemical is removed.

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