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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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Mutation frequency of culture vs. mouse passaged wild type V. cholerae.Wild type cells were grown in LB or passaged through a mouse and plated on (A) rifampicin or (B) trimethopirin to determine the number of resistant colonies. The results show the average mutation frequency of V. cholerae from 5 mice relative to the average mutation frequency from 5 LB grown cultures. The average mutation frequency of the LB grown V. cholerae cultures was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001).
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ppat-1001295-g002: Mutation frequency of culture vs. mouse passaged wild type V. cholerae.Wild type cells were grown in LB or passaged through a mouse and plated on (A) rifampicin or (B) trimethopirin to determine the number of resistant colonies. The results show the average mutation frequency of V. cholerae from 5 mice relative to the average mutation frequency from 5 LB grown cultures. The average mutation frequency of the LB grown V. cholerae cultures was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001).

Mentions: The requirement of BER and mismatch repair (MMR) systems for V. cholerae to efficiently colonize the mouse intestine suggests that V. cholerae experiences increased DNA damage while in the mouse. To address this possibility we measured the mutation frequency of V. cholerae following passage though the mouse as compared to passage in liquid culture. We inoculated five mice and five liquid cultures with the same size inoculums of V. cholerae. The following day we purified bacteria from the mouse intestine (see Materials and Methods). We plated both V. cholerae passaged through the mouse and grown in liquid cultures followed by selection for resistance to two antibiotics we used as an indicator for measuring mutation frequency. The first was a gain of function mutation in rpoB conferring resistance to rifampicin; the second was a loss of function of thyA conferring resistance to trimethoprim. Mutations in rpoB and thyA are well characterized markers for increases in mutation frequency [44], [45], [46]. We found that following passage of V. cholerae through the mouse there was an ∼2 fold increase in rifampicin resistance and ∼2.5 fold increase in trimethoprim resistance compared to the liquid culture grown strains (Figure 2A, B). We sequenced 19 trimethoprim resistance isolates that were passed through the mouse and 20 isolates obtained following growth in liquid culture. We identified 39 unique mutations in thyA (data not shown) suggesting that our results were not influenced by a mutation acquired early on in the procedure. We did not observe a bias in the types of mutation from the two conditions. These results suggest that passage through the mouse results in an increase in mutation rate for V. cholerae suggestive of an increase in DNA damage and the need for repair mechanisms.


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)

Mutation frequency of culture vs. mouse passaged wild type V. cholerae.Wild type cells were grown in LB or passaged through a mouse and plated on (A) rifampicin or (B) trimethopirin to determine the number of resistant colonies. The results show the average mutation frequency of V. cholerae from 5 mice relative to the average mutation frequency from 5 LB grown cultures. The average mutation frequency of the LB grown V. cholerae cultures was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040672&req=5

ppat-1001295-g002: Mutation frequency of culture vs. mouse passaged wild type V. cholerae.Wild type cells were grown in LB or passaged through a mouse and plated on (A) rifampicin or (B) trimethopirin to determine the number of resistant colonies. The results show the average mutation frequency of V. cholerae from 5 mice relative to the average mutation frequency from 5 LB grown cultures. The average mutation frequency of the LB grown V. cholerae cultures was normalized to 1. The error bars reflect the SEM from at least 3 independent experiments (*** p<0.001).
Mentions: The requirement of BER and mismatch repair (MMR) systems for V. cholerae to efficiently colonize the mouse intestine suggests that V. cholerae experiences increased DNA damage while in the mouse. To address this possibility we measured the mutation frequency of V. cholerae following passage though the mouse as compared to passage in liquid culture. We inoculated five mice and five liquid cultures with the same size inoculums of V. cholerae. The following day we purified bacteria from the mouse intestine (see Materials and Methods). We plated both V. cholerae passaged through the mouse and grown in liquid cultures followed by selection for resistance to two antibiotics we used as an indicator for measuring mutation frequency. The first was a gain of function mutation in rpoB conferring resistance to rifampicin; the second was a loss of function of thyA conferring resistance to trimethoprim. Mutations in rpoB and thyA are well characterized markers for increases in mutation frequency [44], [45], [46]. We found that following passage of V. cholerae through the mouse there was an ∼2 fold increase in rifampicin resistance and ∼2.5 fold increase in trimethoprim resistance compared to the liquid culture grown strains (Figure 2A, B). We sequenced 19 trimethoprim resistance isolates that were passed through the mouse and 20 isolates obtained following growth in liquid culture. We identified 39 unique mutations in thyA (data not shown) suggesting that our results were not influenced by a mutation acquired early on in the procedure. We did not observe a bias in the types of mutation from the two conditions. These results suggest that passage through the mouse results in an increase in mutation rate for V. cholerae suggestive of an increase in DNA damage and the need for repair mechanisms.

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