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DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine.

Davies BW, Bogard RW, Dupes NM, Gerstenfeld TA, Simmons LA, Mekalanos JJ - PLoS Pathog. (2011)

Bottom Line: These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract.Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media.Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.

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Related in: MedlinePlus

Growth of RNS/ROS mutants under stress.A. Nitric oxide stress. Exponentially growing cultures of wild type V. cholerae, the ΔhmpA mutant, ΔprxA mutant or ΔprxA ΔhmpA double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, ΔhmpA, ΔprxA and ΔprxA ΔhmpA double mutant treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), ΔhmpA+NO (green), ΔprxA+NO (blue), ΔprxA ΔhmpA+NO (orange), wild type−NO (black), ΔhmpA−NO (purple), ΔprxA−NO (cyan) and ΔprxA ΔhmpA−NO (yellow). The growth of the ΔprxA, ΔhmpA and ΔhmpA ΔprxA mutant is significantly delayed by NO compared to wild type by 435 min (p<0.05). B. H2O2 sensitivity. Wild type (▪) and katB::Tn (▴), perA::Tn (▾) and ahpC::Tn (♦) mutants were plated on increasing concentrations of hydrogen peroxide. Cfu were determined after overnight growth. The average of 3 experiments is shown. C. Superoxide sensitivity. Wild type (▪) and sodA::Tn (▴), ΔsodB (▾) and sodC::Tn (♦) mutants were plated on agar containing increasing concentration of the superoxide generating compound plumbagin. Cfu were determined after overnight growth. The average of 3 experiments is shown. D. Exponentially growing cultures of wild type, the xth::Tn mutant, Δnfo mutant or xth::Tn Δnfo double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, xth::Tn, Δnfo and xth::Tn Δnfo treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), xth::Tn+NO (green), Δnfo+NO (blue), xth::Tn Δnfo+NO (orange), wild type−NO (black), xth::Tn−NO (purple), Δnfo−NO (cyan) and xth::Tn Δnfo−NO (yellow). At 750 min the growth delay of the xth::Tn mutant compared to the wild type in NO is significant (p<0.001) while the growth delay of the Δnfo mutant is not.
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ppat-1001295-g003: Growth of RNS/ROS mutants under stress.A. Nitric oxide stress. Exponentially growing cultures of wild type V. cholerae, the ΔhmpA mutant, ΔprxA mutant or ΔprxA ΔhmpA double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, ΔhmpA, ΔprxA and ΔprxA ΔhmpA double mutant treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), ΔhmpA+NO (green), ΔprxA+NO (blue), ΔprxA ΔhmpA+NO (orange), wild type−NO (black), ΔhmpA−NO (purple), ΔprxA−NO (cyan) and ΔprxA ΔhmpA−NO (yellow). The growth of the ΔprxA, ΔhmpA and ΔhmpA ΔprxA mutant is significantly delayed by NO compared to wild type by 435 min (p<0.05). B. H2O2 sensitivity. Wild type (▪) and katB::Tn (▴), perA::Tn (▾) and ahpC::Tn (♦) mutants were plated on increasing concentrations of hydrogen peroxide. Cfu were determined after overnight growth. The average of 3 experiments is shown. C. Superoxide sensitivity. Wild type (▪) and sodA::Tn (▴), ΔsodB (▾) and sodC::Tn (♦) mutants were plated on agar containing increasing concentration of the superoxide generating compound plumbagin. Cfu were determined after overnight growth. The average of 3 experiments is shown. D. Exponentially growing cultures of wild type, the xth::Tn mutant, Δnfo mutant or xth::Tn Δnfo double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, xth::Tn, Δnfo and xth::Tn Δnfo treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), xth::Tn+NO (green), Δnfo+NO (blue), xth::Tn Δnfo+NO (orange), wild type−NO (black), xth::Tn−NO (purple), Δnfo−NO (cyan) and xth::Tn Δnfo−NO (yellow). At 750 min the growth delay of the xth::Tn mutant compared to the wild type in NO is significant (p<0.001) while the growth delay of the Δnfo mutant is not.

Mentions: RNS, including nitric oxide, have been shown to be powerful antimicrobial agents. The most well studied RNS defense enzyme in bacteria is Hmp, a ferrisiderophore reductase that destroys nitric oxide [47]. V. cholerae carries an hmp homolog, hmpA. Both an hmpA::Tn mutant and a ΔhmpA deletion mutant showed a defect in colonizing the infant mouse intestine (Table 2). Deletion of hmpA delayed V. cholerae growth in the presence of a nitric oxide donor but not in the absence (Figure 3A) consistent with previous observations in other bacteria [20], [48]. This suggests that V.cholerae may encounter deleterious RNS during passage in the mouse. The growth defect of the ΔhmpA mutant in the presence of a nitric oxide donor could be complemented by ectopic expression of hmp from the arabinose inducible plasmid pBAD18 (Figure S1D). In fact, expression of hmp from pBAD18 allowed the ΔhmpA mutant to recover growth more rapidly than the parental strain in the presence of a nitric oxide donor.


DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine.

Davies BW, Bogard RW, Dupes NM, Gerstenfeld TA, Simmons LA, Mekalanos JJ - PLoS Pathog. (2011)

Growth of RNS/ROS mutants under stress.A. Nitric oxide stress. Exponentially growing cultures of wild type V. cholerae, the ΔhmpA mutant, ΔprxA mutant or ΔprxA ΔhmpA double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, ΔhmpA, ΔprxA and ΔprxA ΔhmpA double mutant treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), ΔhmpA+NO (green), ΔprxA+NO (blue), ΔprxA ΔhmpA+NO (orange), wild type−NO (black), ΔhmpA−NO (purple), ΔprxA−NO (cyan) and ΔprxA ΔhmpA−NO (yellow). The growth of the ΔprxA, ΔhmpA and ΔhmpA ΔprxA mutant is significantly delayed by NO compared to wild type by 435 min (p<0.05). B. H2O2 sensitivity. Wild type (▪) and katB::Tn (▴), perA::Tn (▾) and ahpC::Tn (♦) mutants were plated on increasing concentrations of hydrogen peroxide. Cfu were determined after overnight growth. The average of 3 experiments is shown. C. Superoxide sensitivity. Wild type (▪) and sodA::Tn (▴), ΔsodB (▾) and sodC::Tn (♦) mutants were plated on agar containing increasing concentration of the superoxide generating compound plumbagin. Cfu were determined after overnight growth. The average of 3 experiments is shown. D. Exponentially growing cultures of wild type, the xth::Tn mutant, Δnfo mutant or xth::Tn Δnfo double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, xth::Tn, Δnfo and xth::Tn Δnfo treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), xth::Tn+NO (green), Δnfo+NO (blue), xth::Tn Δnfo+NO (orange), wild type−NO (black), xth::Tn−NO (purple), Δnfo−NO (cyan) and xth::Tn Δnfo−NO (yellow). At 750 min the growth delay of the xth::Tn mutant compared to the wild type in NO is significant (p<0.001) while the growth delay of the Δnfo mutant is not.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040672&req=5

ppat-1001295-g003: Growth of RNS/ROS mutants under stress.A. Nitric oxide stress. Exponentially growing cultures of wild type V. cholerae, the ΔhmpA mutant, ΔprxA mutant or ΔprxA ΔhmpA double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, ΔhmpA, ΔprxA and ΔprxA ΔhmpA double mutant treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), ΔhmpA+NO (green), ΔprxA+NO (blue), ΔprxA ΔhmpA+NO (orange), wild type−NO (black), ΔhmpA−NO (purple), ΔprxA−NO (cyan) and ΔprxA ΔhmpA−NO (yellow). The growth of the ΔprxA, ΔhmpA and ΔhmpA ΔprxA mutant is significantly delayed by NO compared to wild type by 435 min (p<0.05). B. H2O2 sensitivity. Wild type (▪) and katB::Tn (▴), perA::Tn (▾) and ahpC::Tn (♦) mutants were plated on increasing concentrations of hydrogen peroxide. Cfu were determined after overnight growth. The average of 3 experiments is shown. C. Superoxide sensitivity. Wild type (▪) and sodA::Tn (▴), ΔsodB (▾) and sodC::Tn (♦) mutants were plated on agar containing increasing concentration of the superoxide generating compound plumbagin. Cfu were determined after overnight growth. The average of 3 experiments is shown. D. Exponentially growing cultures of wild type, the xth::Tn mutant, Δnfo mutant or xth::Tn Δnfo double mutant were grown with or without 1 mM spermine NONOate as a nitric oxide donor. The recovery and growth of each strain was monitored over time. The averages of 3 experiments are shown for each strain. Curves for wild type, xth::Tn, Δnfo and xth::Tn Δnfo treated with spermine NONOate are labeled as “+NO” for clarity. All strains grown in LB without spermine NONOate cluster together and are shown on the plot with the label “All strains−NO”. wild type+NO (red), xth::Tn+NO (green), Δnfo+NO (blue), xth::Tn Δnfo+NO (orange), wild type−NO (black), xth::Tn−NO (purple), Δnfo−NO (cyan) and xth::Tn Δnfo−NO (yellow). At 750 min the growth delay of the xth::Tn mutant compared to the wild type in NO is significant (p<0.001) while the growth delay of the Δnfo mutant is not.
Mentions: RNS, including nitric oxide, have been shown to be powerful antimicrobial agents. The most well studied RNS defense enzyme in bacteria is Hmp, a ferrisiderophore reductase that destroys nitric oxide [47]. V. cholerae carries an hmp homolog, hmpA. Both an hmpA::Tn mutant and a ΔhmpA deletion mutant showed a defect in colonizing the infant mouse intestine (Table 2). Deletion of hmpA delayed V. cholerae growth in the presence of a nitric oxide donor but not in the absence (Figure 3A) consistent with previous observations in other bacteria [20], [48]. This suggests that V.cholerae may encounter deleterious RNS during passage in the mouse. The growth defect of the ΔhmpA mutant in the presence of a nitric oxide donor could be complemented by ectopic expression of hmp from the arabinose inducible plasmid pBAD18 (Figure S1D). In fact, expression of hmp from pBAD18 allowed the ΔhmpA mutant to recover growth more rapidly than the parental strain in the presence of a nitric oxide donor.

Bottom Line: These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract.Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media.Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.

Show MeSH
Related in: MedlinePlus