DNA Damage and Reactive Nitrogen Species are Barriers to Vibrio cholerae Colonization of the Infant Mouse Intestine
[['Maintaining genomic integrity during infection is important for several bacterial pathogens to colonize their hosts .', 'DNA repair defects in Listeria monocytogenes , Salmonella typhimurium , Helicobacter pylori and others leads to decreased or even a complete attenuation of virulence [1]–[6] .', 'While there are several types of DNA repair in bacteria [7] , many of the studies showing a requirement for DNA repair in pathogenesis focus on three pathways: the SOS response , base excision repair , and mismatch repair [1]–[10] .', 'The SOS response is a well studied and conserved stress response in bacteria that is elicited following DNA damage and replication fork arrest [for review [11]] .', 'The SOS response is controlled by positive and negative regulators .', 'RecA positively regulates the SOS response by binding to single-stranded DNA fragments generated by attempted replication past DNA lesions .', 'The RecA/ssDNA nucleoprotein filament induces the auto-cleavage of the negative regulator LexA , a transcriptional repressor .', 'Cleavage of LexA allows for expression of 57 genes in the E . coli SOS regulon including translesion DNA polymerases that are able to replicate DNA past noncoding base lesion , and proteins involved in the inhibition of cell division [for review [11]] .', 'Base excision repair ( BER ) is the most common form of repair for single base damage [for review [7]] .', 'In BER , a DNA N-glycosylase first excises the damaged base from the deoxyribose moiety in the DNA strand creating an abasic site .', 'Class II apurinic/apyrimidinic ( AP ) endonuclease then hydrolyzes the phosphodiester bond immediately 5′ to the abasic site [for review ( Kornberg and Baker 1992 , Friedberg 2005 ) ] .', 'Subsequent actions process this site to prime and repair the abasic site ultimately by DNA synthesis with DNA polymerase I and ligation by DNA ligase .', 'Normal replication can also introduce errors in the form of mismatched DNA base-pairs .', 'These mismatches can lead to permanent mutations after a subsequent round of DNA replication .', 'Mismatch repair ( MMR ) specifically identifies and corrects these base pairing errors increasing the fidelity of the replication pathway nearly ∼1000-fold [for review [7]] .', 'While extensive work has shown the benefit of maintaining genomic integrity for an invading bacterium , there appear to be instances where lapses in genomic fidelity are beneficial for a pathogenic bacterium [12]–[14] .', 'In order to colonize and thrive in a mammalian host , a bacterium must be able to adapt and respond to the conditions and stresses associated its new environment .', 'Genomic mutations support this by allowing current gene products to gain or alter their functions .', "The utility of mutation ( s ) and a pathogen's ability to grow in the human environment has been a source of discussion for several years [13] , [15] .", 'Giraud et al . showed that a high mutation rate was initially beneficial for Escherichia coli to colonize the mouse gut , but this benefit became a liability once adaptation had been reached [12] .', 'Oliver et al . demonstrated that Pseudomonas aeruginosa from chronically infected individuals often has an increased mutation frequency , suggesting an increased mutation rate can be beneficial to P . aeruginosa to allow rapid adaptation to the hostile host environment [14] .', 'Thus depending on the pathogen , the mode and duration of the infection , defects in DNA repair may be detrimental or beneficial to the infecting bacterium .', 'Several studies have indicated that host produced reactive oxygen species ( ROS ) and reactive nitrogen species ( RNS ) cause DNA damage to the invading bacterium [4] , [16] , [17] .', 'Not surprisingly bacteria have several defense mechanisms to detoxify ROS and RNS .', 'Each enzyme detoxifies a specific type of ROS or RNS .', 'For example catalases/peroxidases decompose H2O2 , superoxide dismutases dismutate superoxide and ferrisiderophore reductase removes nitric oxide [18] , [19] .', 'As with certain DNA repair systems , loss of ROS and RNS defenses have been shown to attenuate bacterial pathogens [16] , [20] .', 'Studies supporting the importance of ROS/RNS defenses and DNA repair pathways in bacterial pathogenesis often focus on intracellular pathogens [1] , [2] , [4] .', 'To survive , intracellular pathogens engulfed by phagocytic cells are either able to escape the phagosome or have mechanisms to survive within it .', 'Within the phagosome , captured bacteria may be exposed to host production of ROS and RNS in a host defense response called the oxidative burst .', 'It is hypothesized that the oxidative burst is responsible for the DNA damage experienced by engulfed bacteria [4] , [16] , [17] .', 'Vibrio cholerae is the causative agent of the severe human diarrheal disease cholera .', 'V . cholerae is a non-invasive pathogen that colonizes the small intestine of its host [21] , [22] .', 'As a non-invasive pathogen , V . cholerae is not expected to experience the same types of stresses as intracellular pathogens , such as an oxidative burst .', 'However V . cholerae does pass through several hostile environments as the disease progresses .', 'Immediately following ingestion , V . cholerae is exposed to the exceptionally antagonistic environment of the stomach where the pH of gastric acid can reach as low as 1 [23] , [24] .', 'Furthermore , nitrite from both food sources and the salivary nitrite cycle can enter the stomach creating acidified nitrite [25] , [26] , [27] .', 'Acidified nitrite has potent antimicrobial effects on gut pathogens [28] , [29] , [30] , [31] .', 'These studies show that the viability of several pathogenic bacteria decreases rapidly under acidified nitrite conditions .', 'Furthermore , nitrates , which can also be found in the stomach , have been shown to modify of gene expression reducing acid tolerance [32] .', 'The antimicrobial effects of acidified nitrite are thought to be due to the generation of deleterious RNS [33] .', 'However , with the exception of a few studies [34] , [35] , [36] , the points of action of these RNS as well as the bacterial determinants required for protection against them have remained largely unexamined .', 'After traversing the stomach V . cholerae faces several innate host defenses in the intestine including bile , lysozyme , small antimicrobial peptides and complement [37] .', 'Thus , V . cholerae must overcome several barriers during infection that have the potential to cause DNA damage through a direct or indirect mechanism .', 'We report here that V . cholerae strain C6706 experiences increased DNA damage during passage through the murine gastrointestinal track .', 'We demonstrate that increased genomic stress is a potential barrier to host colonization by V . cholerae .', 'We found that two important DNA repair pathways are necessary for V . cholerae to efficiently colonize the infant mouse intestine .', 'Furthermore , we show that defense against RNS is also necessary for V . cholerae to colonize the infant mouse .', 'In doing so we identify a novel protein required for defense against RNS in pathogenic bacteria .', 'In vitro we show that all our colonization defective DNA repair and RNS defense mutants share a common sensitivity to acidified nitrite and we further show that neutralizing stomach acid rescues intestinal colonization defect of these mutants .'], ['To determine if V . cholerae requires defenses against DNA damage during colonization , we tested a series of transposon mutants that contained insertions in different steps in three important DNA repair pathways for their ability to colonize the infant mouse intestine in competition with the wild type strain .', 'These pathways were nucleotide excision repair ( NER ) , base excision repair ( BER ) and mismatch repair ( MMR ) ( Table 1 ) .', 'While the SOS response is an important contributor to genomic integrity we did not test a requirement for SOS since Quiones et al . previously showed that SOS activation is not required for intestinal cholera toxin production or colonization [38] .', 'We used uvrA as a representative gene required for NER since uvrA is obligatory for NER .', 'We found no difference in the ability of the uvrA::Tn containing strain to colonize the infant mouse relative to the parental strain suggesting that NER is dispensable for V . cholerae pathogeneis ( Table 1 ) .', 'Apurinic/apyrimidininc ( AP ) endonucleases are critical in BER .', 'BER has been most well studied in E . coli .', 'E . coli encodes two class II AP endonucleases , Xth [endo II ( endo VI ) ] and Nfo ( endo IV ) .', 'In E . coli Xth is responsible for ∼90% of the AP endonuclease activity in the cell [39] , [40] .', 'Few phenotypes have been attributed solely to Nfo activity but Nfo is known to contribute to BER [41] .', 'V . cholerae carries close homologs of both Xth and Nfo ( VC1860 and VC2360 respectively ) .', 'Interestingly we found that the xth::Tn mutant was not defective in intestinal colonization however the nfo::Tn mutant showed a defect in colonization compared to the parental strains ( Table 1 ) .', 'We created a clean deletion of nfo ( Δnfo ) in V . cholerae and found this mutant also had a colonization defect .', 'In E . coli deletion of xth and nfo leads to a more profound defect in DNA repair than either single mutant .', 'Consistent with this observation , we found that an xth::Tn Δnfo double mutant showed a ∼10-fold defect in colonization that appears slightly greater than the ∼5-fold defect in colonization of the Δnfo mutant alone ( Table 1 ) , although this difference is not statistically significant for this number of replicates tested ( p>0 . 05 ) .', 'Thus , these results suggest that BER is important for V . cholerae to colonize the infant mouse intestine when in competition .', 'These results also show a critical function for Nfo in survival , which has not been apparent under laboratory conditions .', 'Loss of mismatch repair function has been shown to be either beneficial or detrimental depending on the pathogen studied [12] , [13] , [14] , [15] .', 'We found that a transposon mutant in mutS , which encodes the gene product that initially binds to a mismatch , resulted in a decrease in colonization efficiency ( Table 1 ) .', 'We constructed a clean deletion of mutS ( ΔmutS ) to ensure the defect was not due to the transposon .', 'We found that the clean deletion of mutS was also attenuated in its ability to colonize the intestine suggesting that mismatch repair or at least MutS is important for V . cholerae pathogenesis ( Table 1 ) .', 'We also found that a second clean deletion of mutS showed a similar competitive index defect ( CI\u200a=\u200a0 . 18±0 . 03 ) suggesting that the colonization defect was not due to mutations in the first mutS clean deletion strain .', 'We noted that the mutS transposon mutant was more defective than its clean deletion counterpart ( Table 1 ) .', 'This difference may be due to a polar effect of the transposon or mutations acquired by the mutS::Tn strain during outgrowth of the original isolate .', 'We also tested the colonization proficiency of a Δnfo ΔmutS double mutant and found that the colonization defect of this double mutant appears slightly greater than either the Δnfo or ΔmutS mutant alone ( Table', '1 ) although this difference is not statistically significant for this number of replicates tested ( p>0 . 05 ) .', 'The requirement of BER and MMR for V . cholerae to efficiently colonize the infant mouse intestine suggests that V . cholerae experiences DNA damage in the mouse , and that a reduced ability to repair such damage is detrimental for V . cholerae pathogenesis .', '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 .', 'Loss of Xth activity in E . coli renders the strain sensitive to hydrogen peroxide ( H2O2 ) [43] .', 'We found that our xth::Tn strain was also sensitive to H2O2 ( Figure 1B ) .', 'Loss of nfo activity alone does not greatly sensitize E . coli to H2O2 but loss of xth and nfo creates a strain with increased sensitivity to H2O2 [43] .', 'We found a similar effect in V . cholerae where the xth::Tn Δnfo strain was much more sensitive to H2O2 then the xth::Tn mutant alone ( Figure 1B ) .', 'Furthermore , high level expression of nfo from a plasmid complemented the H2O2 sensitivity of the xth::Tn Δnfo mutant ( Figure S1C ) .', 'These results suggest that V . cholerae Nfo acts like its E . coli homolog .', '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 .', 'We have identified two DNA repair mechanisms required by V . cholerae to efficiently colonize the infant mouse , and have shown that V . cholerae passaged through a mouse has an increased mutation frequency .', 'Thus , we sought to identify potential causes of DNA damage for V . cholerae while in the mouse to understand the requirement for BER and MMR in the mouse .', 'A major source of DNA damage for intracellular pathogens is from host produced ROS and RNS .', 'While V . cholerae is a non-invasive pathogen we considered that it still may experience ROS and RNS at some point during infection .', 'We used a genetic approach to determine if ROS/RNS affected V . cholerae colonization and if so what type ( s ) of ROS/RNS were most important during this encounter .', 'Bacteria have several enzymes to detoxify ROS/RNS .', 'Each enzyme detoxifies a specific type of ROS or RNS [for review see [18] , [19] .', 'For example catalases/peroxidases decompose H2O2 , superoxide dismutases remove superoxide and ferrisiderophore reductases remove nitric oxide .', 'Bacteria can contain multiple proteins capable of dealing with one type of stress .', 'V . cholerae possesses two catalases/peroxidases ( KatB/PerA ) and one alkyl hydroperoxide reductase ( AhpC ) , three superoxide dismutases ( SodA/B/C ) but only one ferrisiderophore reductase ( HmpA ) .', 'We tested mutants defective for each of these different types of defense enzymes to identify the type ( s ) of radicals that may be damaging V . cholerae in the mouse ( Table 2 ) .', '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 .', 'After testing all previously predicted antioxidant enzymes we began to mine the V . cholerae genome for additional putative antioxidant enzymes .', 'We began by searching for putative proteins that belonged to large antioxidant families .', 'Enzymes , such as AhpC , belong to the Peroxiredoxin ( PRX ) family .', 'Searching for peroxiredoxin family proteins yielded a putative defense enzyme we have called PrxA ( VC2637 ) .', 'PrxA , classified is a peroxiredoxin-5 family protein , is found in several pathogenic bacteria and is a distant homolog of a macrophage peroxynitrite detoxification protein [49] .', 'Deletion of prxA did not effect V . cholerae growth in LB alone but significantly delayed V . cholerae growth in the presence of a nitric oxide donor ( Figure 3A ) .', 'Furthermore , both the prxA::Tn mutant and the ΔprxA allele we constructed caused a decrease in the ability of V . cholerae to colonize the infant mouse in competition assays ( Table 2 ) .', 'The growth defect of the ΔprxA mutant in the presence of a nitric oxide donor could be complemented by ectopic expression of prxA from the arabinose inducible plasmid pBAD18 ( Figure S1E ) .', 'The discovery of a new gene required for both defense against RNS and efficient colonization of the infant mouse further supports our findings that V . cholerae may be exposed to RNS during passage though the mouse .', 'We tested the sensitivity of a ΔprxA ΔhmpA double mutant and found that the growth of the double mutant in the presence of a nitric oxide donor was even more delayed than either the ΔprxA or ΔhmpA single mutant alone ( Figure 3A ) .', 'We also tested the colonization efficiency of a ΔprxA ΔhmpA ( Table', '2 ) and found that it was not significantly less than the ΔprxA mutant alone ( p>0 . 05 ) .', 'Thus hmpA and prxA are both important for colonization but the effects were not additive .', 'We asked if defects in ROS defense also affect V . cholerae colonization .', 'Disruption of ahpC , katB , perA , sodA or sodC did not affect the ability of V . cholerae to colonize the infant mouse and these deficiencies did not affect the ability of V . cholerae to colonize the infant mouse in competition experiments ( Table 2 ) .', 'We did not test the sodB::Tn mutant since both it and a ΔsodB deletion strain we constructed had a decreased plating efficiency and grew very poorly compared to the parental strain ( Figure S1F ) .', 'Thus , while SodB appears to be important for growth of V . cholerae under laboratory conditions we did not pursue the sodB mutant in mouse experiments .', 'Interestingly , of ahpC , katB and perA only disruption of perA sensitized V . cholerae to H2O2 in vitro ( Figure 3B ) .', 'Furthermore , of strains disrupted individually for sodA , sodB and sodC only disruption of sodB sensitized V . cholerae to the superoxide generating compound plumbagin ( Figure 3C ) .', 'We also tested the ΔprxA mutant but found that it did not show increased sensitivity to either H2O2 or plumbagin ( Figure S2A and data not shown ) .', 'It is possible that some of these known ROS defense enzymes overlap in function masking the effects of a deficiency in any one gene in vitro or in mouse studies .', 'For other bacterial pathogens and symbionts deletion of several or all catalases and superoxide dismutases has been required before a strong effect on virulence or symbiosis was observed [50] , [51] , [52] .', 'Currently , the results from our analysis suggest RNS may pose a significant barrier to V . cholerae in colonizing the infant mouse .', 'ROS may also play a role , however their effect is not immediately evident in our analysis .', 'XthA appears to be more important than Nfo in protecting V . cholerae against environmental stress in vitro ( Figure 1B ) , yet Nfo appears to be more important for colonization of the small intestine ( Table 1 ) .', 'The requirement for ΔhmpA and ΔprxA for efficient intestinal colonization lead us to ask if Nfo was required for defense against nitric oxide .', 'We monitored the growth of the xth::Tn , Δnfo and xth::Tn Δnfo mutants in the presence of a nitric oxide donor .', 'We found that , at least in vitro , xth:Tn was more important than Nfo for protection against nitric oxide ( Figure 3D ) .', 'We also found that the double mutant was again more sensitive to the stress than either single mutant alone ( Figure 3D ) .', 'Our results led us to ask if our DNA repair and RNS defense defective V . cholerae mutants were sensitive to any host defenses .', 'The intestine has several innate defenses [37] .', 'We tested many of these defenses including lysozyme , phospholipase , antimicrobial peptides , complement , bile , changes in osmolarity and pH , however , we did not observe any difference in sensitivity between the parental and the mutant strains ( data not shown ) .', 'RNS have been shown to be generated by macrophages to kill phagocytised bacteria [reviewed in [53]] .', 'The RNS from macrophages is generated by an inducible nitric oxide synthase ( iNOS ) .', 'Inhibition of iNOS activity has been shown to rescue the virulence defects in hmp mutant strains of Salmonella enterica serovar typhimurium [20] .', 'However , our hmpA::Tn V . cholerae mutant showed no difference in it ability to colonize the intestine of a wild type or isogenic iNOS−/−infant mouse ( Table S1 ) .', 'Thus , our results suggest that the colonization defect of the DNA repair and RNS defense mutants may occur before V . cholerae is exposed to the host defenses found in the small intestine .', 'In the stomach V . cholerae is exposed to low pH in combination with µM amounts of nitrite from ingested food and the salivary nitrite cycle [25] , [26] , [27] .', 'Acidified nitrite produces a variety of toxic RNS .', 'We quantified the amount of nitrite in the infant mouse stomach using the Griess reaction and found that it was 20 . 0±0 . 7 µM , which is similar to that of humans [26] .', 'The pH range of human gastric juice is reported as 1–3 [23] , [24] .', 'We determined the pH of the infant mouse stomach to be 4 . 5±0 . 1 using a fluorescent pH sensitive dye .', 'This measurement is conservative and the pH of the infant mouse gastric juice may be even less ( see Materials and Methods ) .', 'Thus , the infant mouse stomach is sufficiently acidic to promote the formation of acidified nitrite .', '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 .', 'Only the growth of the ΔmutS strain was unaffected at 400 µM .', 'We considered that while MutS may not be required for survival of acidified nitrite during this time course it may be required to prevent acidified nitrite induced mutations in V . cholerae that are detrimental for colonization .', 'We grew V . cholerae in LB at pH 5 . 5 over night in the presence or absence of 600 µM nitrite and then plated for rifampicin resistant colonies .', 'We found that V . cholerae grown in the presence of nitrite had a greater than 10-fold increase in mutation frequency compared to the media only control ( Figure 4C ) .', 'Loss of MutS then increased the mutation frequency of V . cholerae in nitrite at pH 5 . 5 ∼ an additional 5-fold ( Figure 4C ) .', 'Thus , MutS may be important to prevent acidified nitrite induced mutations that could impair the ability of V . cholerae to colonize the infant mouse .', 'To further test this possibility we created a ΔhmpA ΔmutS double mutant and tested its colonization proficiency ( Table 2 ) .', 'Interestingly , the colonization defect of the ΔhmpA ΔmutS double mutant was not significantly different than either of the single mutants alone ( p>0 . 05 ) .', 'This result may suggest that HmpA and MutS may share a similar defense pathway in the infant mouse .', 'Additionally , E . coli MutS can recognize an O6-methyl-dG:dC base pair , a mutation which can occur by alkylation of G bases [55] .', 'Therefore it is possible that MutS may also be important for protection again some type of alkylation that occurs in the mouse stomach .', 'If acidified nitrite produces RNS that damaged V . cholerae DNA , we reasoned that we should be able to detect increased intracellular radical formation in V . cholerae following nitrite treatment .', 'We grew V . cholerae at pH 5 . 5 with increasing amounts of nitrite and assayed for radical formation using 2′ , 7′-dichlorodihydrofluorescein diacetate ( H2DCFDA ) .', 'H2DCFDA is a cell permeable dye that fluoresces after reacting with RNS and ROS species .', 'H2DCFDA reacts with several ROS and RNS including hydrogen peroxide , nitric oxide and peroxynitrite [56] , [57] , [58] .', 'We found that H2DCFDA fluorescence correlated with increasing nitrite concentration at pH 5 . 5 indicating an increase in intracellular radicals ( Figure 4D ) .', 'For comparison , we found that H2DCFDA fluorescence did not increase over the concentrations of nitrate examined at pH 7 . 0 .', 'These results further support the requirement for low pH to induce radical formation from nitrite sources ( Figure 4D ) .', 'Our results suggested that V . cholerae may experience DNA damage as it passes through the stomach .', 'If so , we were curious if a colonization defect could be observed at an early time point after inoculation .', 'We repeated the competitive colonization assays testing the ΔmutS , Δnfo , ΔprxA and ΔhmpA mutants .', 'We found that at 3 hours post inoculation each mutant strain already showed a 50–60% colonization defect when co-inoculated with the wild type ( Table S2 ) though this defect was not as great as the 5–20% defects reported in Table 1 .', 'This suggests that a defect of the mutant strains is detrimental for colonization early after inoculation .', 'Angelichio et al . [59] reported that V . cholerae populations in the small intestine do not show a significant increase in number until between 10–24 h post inoculations .', 'These results suggest that the effects of the DNA damage are ∼50% detrimental at the earliest stages of infection and become more apparent as the bacteria replicate to high numbers in the intestine .', 'Such a conclusion is consistent with the concept of damage occurring primarily in the stomach .', 'Our results support a relationship between acidified nitrite sensitivity and colonization defects in our RNS and DNA repair .', 'We reasoned that if acidified nitrite in the stomach was responsible for the colonization defects of our mutants then neutralizing stomach acid in the mouse should relieve , at least in part , the observed colonization defects .', 'We used sodium bicarbonate to neutralize the mouse stomach acid .', 'When we inoculated infant mice with our DNA repair and RNS defense defective mutants in the presence of sodium bicarbonate all four mutant strains ( Δnfo , ΔmutS , ΔprxA and ΔhmpA ) showed significant improvement in their ability to colonize the intestine in competition with the parental strain ( Table 3 ) .', 'In fact the colonization defect of the ΔmutS and Δnfo mutant was completely rescued .', 'The colonization defect of the ΔprxA was restored to near wild type levels .', 'The colonization defect of the ΔhmpA mutant was partially rescued although this difference is not statistically significant for the number of replicates tested ( p>0 . 05 ) .', 'The nitrite concentration of the infant mouse stomach after sodium bicarbonate treatment remained nearly unchanged at 20 . 3±0 . 8 µM .'], ['We have shown that V . cholerae must defend against DNA damage to efficiently colonize the infant mouse intestine and that such damage likely occurs early during infection as V . cholerae enters the stomach .', 'We have demonstrated that V . cholerae specifically requires BER and MMR pathways to efficiently colonize the infant mouse intestine .', 'Furthermore , we have identified one previously known and one novel RNS defense protein that facilitates intestinal colonization of the infant mouse .', 'These DNA repair and RNS defense proteins were also required for V . cholerae to grow or maintain genomic fidelity in the presence of acidified nitrite .', 'Furthermore the colonization defects of each mutant could be partially or fully complemented by neutralizing stomach acid suggesting that RNS defense and DNA repair share a common defensive role in the mouse .', 'V . cholerae has been shown to be very sensitive to low pH [54] .', 'For this reason , human volunteers have their stomach contents neutralized to promote experimental V . cholerae infection as is done with live attenuated vaccine studies [60] .', 'In the recently developed infant rabbit model for cholera , stomach acid is also neutralized and cimetidine is administered to prevent re-acidification in order for V . cholerae to colonize the infant rabbit intestine [61] .', 'However , our DNA repair and RNS defense mutants did not show increased sensitivity to low pH compared to the parent strain .', 'Agreeing with our observations a large screen used to identify genes necessary for colonization and tolerating low pH in V . cholerae did not identify any of the genes we reported here for influencing colonization [62] .', 'While low pH of the stomach alone is undoubtedly detrimental towards V . cholerae our results suggest that neutralizing stomach acid may also be important to prevent RNS formation by acidified nitrite .', 'We propose that the defect in colonization of the DNA repair and RNS defense mutants is due to RNS formation .', 'Acidified nitrite is present in the stomach where gastric juice interacts from nitrite sources from the diet or salivary nitrite pathway .', 'The chemistry of acidified nitrite is known to produce several potentially deadly radicals ( 1 ) .', '( 1 ) Both NO• and NO2• can directly attack cellular macromolecules , but they can also interact with other radicals to form further species such as peroxynitrite and hydroxyl acids .', 'From equation ( 1 ) we can see that a key factor in this proces
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['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 .']
['Studies on intracellular bacterial pathogens have shown the need for maintaining genomic fidelity to promote colonization .', 'Loss of DNA repair functions often leads to attenuation and rapid clearing of the invading pathogen .', 'However , for some pathogens , an increased mutation rate has been shown to be beneficial for promoting host colonization , presumably by allowing the pathogen to rapidly adapt to adverse host conditions .', 'We asked if the non-invasive pathogen V . cholerae experienced increased DNA damage during infection and if so , how the increased damage influenced host colonization and from where the source of the damage was derived .', 'Our results demonstrate that V . cholerae experiences increased DNA damage during infection in the infant mouse model and that loss of ability to repair this damage results in attenuation of virulence .', 'We specifically show that V . cholerae requires both base excision repair and mismatch repair for efficient intestinal colonization .', 'Furthermore , we present evidence that the source of the DNA damage is derived from reactive nitrogen species ( RNS ) formed by acidified nitrite in the mouse gut and in doing so we identify a new RNS defense protein found in V . cholerae and several other pathogenic bacteria .']
['molecular biology/dna repair', 'genetics and genomics/disease models', 'infectious diseases/bacterial infections', 'microbiology/cellular microbiology and pathogenesis', 'microbiology/medical microbiology', 'infectious diseases/gastrointestinal infections']