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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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Effect of nitrite on growth of wild type and mutant V. cholerae strains.A/B. Exponentially growing cultures of wild type and ΔmutS, Δnfo, ΔprxA, ΔhmpA and Δnfo xth::Tn mutants were grown in LB buffered at pH 5.5 in the absence (A) or presence (B) of 400 µM sodium nitirite. The average of three experiments is shown for each strain. Wild type (black squares), ΔmutS (blue triangles), Δnfo (orange circles), ΔprxA (green inverted triangle), ΔhmpA (red diamond) and Δnfo xth::Tn (yellow open square). C. Mutation frequency as measured by rifampicin resistant colony formation frequency from wild type and ΔmutS mutant cultures grown at pH 5.5 in the presence or absence of 600 µM sodium nitrite. The average mutation frequency of the wild type grown in the absence sodium nitrite was normalized to 1 (* p<0.05, ** p<0.01 compared to wild type) D. Intracellular RNS production following nitrite treatment. Wild type cultures were grown at pH 7.0 or 5.5 plus 0, 0.5, 1.0, 5.0, or 10.0 mM sodium nitrite. After washing cells were exposed to the radical binding dye H2DCFDA. After removal of media, cells were lysed and H2DCFDA fluorescence was measured. The average of at least 3 independent experiments is shown with error bars representing the SEM (* p<0.05, ** p<0.01).
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ppat-1001295-g004: Effect of nitrite on growth of wild type and mutant V. cholerae strains.A/B. Exponentially growing cultures of wild type and ΔmutS, Δnfo, ΔprxA, ΔhmpA and Δnfo xth::Tn mutants were grown in LB buffered at pH 5.5 in the absence (A) or presence (B) of 400 µM sodium nitirite. The average of three experiments is shown for each strain. Wild type (black squares), ΔmutS (blue triangles), Δnfo (orange circles), ΔprxA (green inverted triangle), ΔhmpA (red diamond) and Δnfo xth::Tn (yellow open square). C. Mutation frequency as measured by rifampicin resistant colony formation frequency from wild type and ΔmutS mutant cultures grown at pH 5.5 in the presence or absence of 600 µM sodium nitrite. The average mutation frequency of the wild type grown in the absence sodium nitrite was normalized to 1 (* p<0.05, ** p<0.01 compared to wild type) D. Intracellular RNS production following nitrite treatment. Wild type cultures were grown at pH 7.0 or 5.5 plus 0, 0.5, 1.0, 5.0, or 10.0 mM sodium nitrite. After washing cells were exposed to the radical binding dye H2DCFDA. After removal of media, cells were lysed and H2DCFDA fluorescence was measured. The average of at least 3 independent experiments is shown with error bars representing the SEM (* p<0.05, ** p<0.01).

Mentions: At pH 3 in rich medium we found that V. cholerae had a greater than 99.9% decrease in survival in less than 1 minute (data not shown) agreeing with similar work examining V. cholerae acid tolerance [54]. We did not find a difference in survival between the parental and mutant strains at low pH (1–4) levels (data not shown). We gradually increased pH to identify the lowest level at which V. cholerae could grow. At pH 5.5 V. cholerae and the DNA repair and RNS defense mutants grew with identical kinetics (Figure 4A). We titrated nitrite into the growth medium and found that nearly all the mutant strains showed a growth defect compared to the wild type at pH 5.5 in the presence of 400 µM nitrite (Figure 4B). No differences in growth between wild type and mutant strains were observed at pH 7.0 with or without 400 µM nitrite (Figure S3A, B). Not only did low pH and nitrite slow the growth of our mutants but the ΔhmpA, ΔprxA, Δnfo and xth::Tn Δnfo mutants began to show a decrease in optical density after longer exposure (Figure 4B) suggesting the cells were lysing.


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)

Effect of nitrite on growth of wild type and mutant V. cholerae strains.A/B. Exponentially growing cultures of wild type and ΔmutS, Δnfo, ΔprxA, ΔhmpA and Δnfo xth::Tn mutants were grown in LB buffered at pH 5.5 in the absence (A) or presence (B) of 400 µM sodium nitirite. The average of three experiments is shown for each strain. Wild type (black squares), ΔmutS (blue triangles), Δnfo (orange circles), ΔprxA (green inverted triangle), ΔhmpA (red diamond) and Δnfo xth::Tn (yellow open square). C. Mutation frequency as measured by rifampicin resistant colony formation frequency from wild type and ΔmutS mutant cultures grown at pH 5.5 in the presence or absence of 600 µM sodium nitrite. The average mutation frequency of the wild type grown in the absence sodium nitrite was normalized to 1 (* p<0.05, ** p<0.01 compared to wild type) D. Intracellular RNS production following nitrite treatment. Wild type cultures were grown at pH 7.0 or 5.5 plus 0, 0.5, 1.0, 5.0, or 10.0 mM sodium nitrite. After washing cells were exposed to the radical binding dye H2DCFDA. After removal of media, cells were lysed and H2DCFDA fluorescence was measured. The average of at least 3 independent experiments is shown with error bars representing the SEM (* p<0.05, ** p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040672&req=5

ppat-1001295-g004: Effect of nitrite on growth of wild type and mutant V. cholerae strains.A/B. Exponentially growing cultures of wild type and ΔmutS, Δnfo, ΔprxA, ΔhmpA and Δnfo xth::Tn mutants were grown in LB buffered at pH 5.5 in the absence (A) or presence (B) of 400 µM sodium nitirite. The average of three experiments is shown for each strain. Wild type (black squares), ΔmutS (blue triangles), Δnfo (orange circles), ΔprxA (green inverted triangle), ΔhmpA (red diamond) and Δnfo xth::Tn (yellow open square). C. Mutation frequency as measured by rifampicin resistant colony formation frequency from wild type and ΔmutS mutant cultures grown at pH 5.5 in the presence or absence of 600 µM sodium nitrite. The average mutation frequency of the wild type grown in the absence sodium nitrite was normalized to 1 (* p<0.05, ** p<0.01 compared to wild type) D. Intracellular RNS production following nitrite treatment. Wild type cultures were grown at pH 7.0 or 5.5 plus 0, 0.5, 1.0, 5.0, or 10.0 mM sodium nitrite. After washing cells were exposed to the radical binding dye H2DCFDA. After removal of media, cells were lysed and H2DCFDA fluorescence was measured. The average of at least 3 independent experiments is shown with error bars representing the SEM (* p<0.05, ** p<0.01).
Mentions: At pH 3 in rich medium we found that V. cholerae had a greater than 99.9% decrease in survival in less than 1 minute (data not shown) agreeing with similar work examining V. cholerae acid tolerance [54]. We did not find a difference in survival between the parental and mutant strains at low pH (1–4) levels (data not shown). We gradually increased pH to identify the lowest level at which V. cholerae could grow. At pH 5.5 V. cholerae and the DNA repair and RNS defense mutants grew with identical kinetics (Figure 4A). We titrated nitrite into the growth medium and found that nearly all the mutant strains showed a growth defect compared to the wild type at pH 5.5 in the presence of 400 µM nitrite (Figure 4B). No differences in growth between wild type and mutant strains were observed at pH 7.0 with or without 400 µM nitrite (Figure S3A, B). Not only did low pH and nitrite slow the growth of our mutants but the ΔhmpA, ΔprxA, Δnfo and xth::Tn Δnfo mutants began to show a decrease in optical density after longer exposure (Figure 4B) suggesting the cells were lysing.

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