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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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Phenotypes of V. cholerae DNA repair mutants.A. Mutation frequency. The number of ΔmutS rifampicin resistant colonies relative to wild type colonies is shown. The number of V. cholerae colonies was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001). B. Hydrogen peroxide sensitivity. The sensitivity of wild type (▪), xth::Tn (▾), Δnfo (▴) and xth::Tn Δnfo (♦) strains to increasing concentrations of hydrogen peroxide are shown ± SEM from at least 3 independent experiments. The xth::Tn mutant is statistically different from the wild type and the Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001). The xth::Tn Δnfo strain is significantly different from the wild type, xth::Tn and Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001).
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ppat-1001295-g001: Phenotypes of V. cholerae DNA repair mutants.A. Mutation frequency. The number of ΔmutS rifampicin resistant colonies relative to wild type colonies is shown. The number of V. cholerae colonies was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001). B. Hydrogen peroxide sensitivity. The sensitivity of wild type (▪), xth::Tn (▾), Δnfo (▴) and xth::Tn Δnfo (♦) strains to increasing concentrations of hydrogen peroxide are shown ± SEM from at least 3 independent experiments. The xth::Tn mutant is statistically different from the wild type and the Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001). The xth::Tn Δnfo strain is significantly different from the wild type, xth::Tn and Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001).

Mentions: V. cholerae genes encoding Xth, Nfo and MutS were identified based on sequence similarity with their well-studied E. coli homologs. To ensure the V. cholerae homologs possessed their predicted functions we tested our mutant strains for the well characterized phenotypes described in other bacterial systems. Loss of mismatch repair causes an increase in mutation rate often referred to as a mutator phenotype [42]. We found that our ΔmutS mutant had a significantly increased mutation frequency compared with the wild type control (Figure 1A). The wild type phenotype could be restored by expression of mutS from a plasmid but not by the plasmid itself (Figure S1B). This result indicates that MutS in V. cholerae shares the same activity as its other well studied bacterial homologs in the repair of DNA replication errors.


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)

Phenotypes of V. cholerae DNA repair mutants.A. Mutation frequency. The number of ΔmutS rifampicin resistant colonies relative to wild type colonies is shown. The number of V. cholerae colonies was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001). B. Hydrogen peroxide sensitivity. The sensitivity of wild type (▪), xth::Tn (▾), Δnfo (▴) and xth::Tn Δnfo (♦) strains to increasing concentrations of hydrogen peroxide are shown ± SEM from at least 3 independent experiments. The xth::Tn mutant is statistically different from the wild type and the Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001). The xth::Tn Δnfo strain is significantly different from the wild type, xth::Tn and Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040672&req=5

ppat-1001295-g001: Phenotypes of V. cholerae DNA repair mutants.A. Mutation frequency. The number of ΔmutS rifampicin resistant colonies relative to wild type colonies is shown. The number of V. cholerae colonies was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001). B. Hydrogen peroxide sensitivity. The sensitivity of wild type (▪), xth::Tn (▾), Δnfo (▴) and xth::Tn Δnfo (♦) strains to increasing concentrations of hydrogen peroxide are shown ± SEM from at least 3 independent experiments. The xth::Tn mutant is statistically different from the wild type and the Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001). The xth::Tn Δnfo strain is significantly different from the wild type, xth::Tn and Δnfo mutant at 100µM and 200µM H2O2 (*** p<0.001).
Mentions: V. cholerae genes encoding Xth, Nfo and MutS were identified based on sequence similarity with their well-studied E. coli homologs. To ensure the V. cholerae homologs possessed their predicted functions we tested our mutant strains for the well characterized phenotypes described in other bacterial systems. Loss of mismatch repair causes an increase in mutation rate often referred to as a mutator phenotype [42]. We found that our ΔmutS mutant had a significantly increased mutation frequency compared with the wild type control (Figure 1A). The wild type phenotype could be restored by expression of mutS from a plasmid but not by the plasmid itself (Figure S1B). This result indicates that MutS in V. cholerae shares the same activity as its other well studied bacterial homologs in the repair of DNA replication errors.

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