Despite a wealth of research on the virulence factors that allow V. cholerae to cause disease, relatively little is known about the challenges that V. cholerae encounters during infection of the intestines and how it senses and overcomes them. In this study, we have identified how V. cholerae senses and responds to NO, a common challenge for intestinal pathogens. We have further demonstrated that one of the NO detoxification genes, hmpA, and its transcriptional activator NorR are critical for sustained colonization of the intestines of mice.
Previous bioinformatic analysis led to the identification of a remarkably limited repertoire of NO-related genes found in the V
genome, even compared to highly related Vibrio
). Using reporter assays, we demonstrated that the expression of two of these genes, nnrS
and the flavohemoglobin-encoding gene hmpA
, is highly inducible by the addition of NO to microaerobically growing cells. This upregulation was dependent on the σ54
-dependent transcriptional regulator NorR (23
). Growth curve analysis demonstrated that these genes are essential for NO resistance in vitro
. Intriguingly, a strain of V
lacking both hmpA
was the most attenuated for growth in the presence of NO; concomitantly, deletion of norR
resulted in a nearly equivalent growth defect in the presence of NO. These data demonstrated that HmpA is the principal detoxifier of NO but that NnrS may play an auxiliary role. The only study of NnrS published to date identified it as a heme- and copper-containing membrane protein in Rhodobacter sphaeroides
). However, nnrS
homologs are found in the genomes of human pathogens such as Pseudomonas
, and Neisseria
, suggesting that it may play NO detoxification roles in a variety of infectious settings. The exact function of NnrS is an area of current investigation in our laboratory.
The role of NO detoxification genes in V
pathogenesis has been examined in an infant mouse model in which bacteria are allowed to colonize the intestines for 24 h (22
). After this brief period, there was a moderate colonization defect in the hmpA
mutant attributed to the low pH of the stomach. We were interested in whether NO resistance could be important in colonization of the intestine over a time period resembling that of a human infection. Interestingly, we found that the importance of HmpA was much greater than previously thought; there were virtually no hmpA
mutants recovered from fecal samples or small intestinal homogenates after 7 days. This defect was partially due to iNOS-derived stress, as the colonization defect was partially mitigated in iNOS−/−
mice at 7 days. The remaining defect is not likely to be due to stomach acidity because the mice were administered bicarbonate prior to inoculation. Mice and humans possess two other NOS isoforms, neuronal NOS and endothelial NOS (31
), which may also account for some of the defect that persists in iNOS−/−
We were surprised to discover the effects of the nnrS mutation on colonization. Although the hmpA nnrS double mutant was severely inhibited in vitro, this mutant fared no better in iNOS−/− mice than in wild-type mice. Furthermore, the nnrS single mutant slightly outcompeted wild-type V. cholerae in wild-type mice but was attenuated in iNOS−/− mice. It is difficult to interpret these data, given the unknown function of NnrS, but we hypothesize that the complex metabolism of RNS results in the buildup of detrimental chemical products in some contexts. Furthermore, an acknowledged disadvantage of competition studies is that a defect in the nnrS mutant may be complemented in trans by the wild-type coinoculated strain. Future studies may address this possibility. Given the in vitro importance of NnrS, however, we speculate that there are infectious settings in which NnrS is critical to the survival of V. cholerae. In addition, we were surprised to find that the hmpA nnrS double mutant had a far more severe colonization defect than the norR mutant in wild-type mice (), since NorR is absolutely required for the upregulation of hmpA and nnrS in response to NO (). One possible explanation for the discrepancy between the colonization defects is that baseline transcription of hmpA and nnrS in the norR deletion mutant, however low, is sufficient to detoxify a significant proportion of the NO stress found in vivo. Alternatively, signals other than NO, and thus regulators other than NorR, might cause the upregulation of hmpA and nnrS in vivo. This could allow better colonization efficiency than when hmpA and nnrS are deleted entirely. Our laboratory is currently working to find these alternative signals and regulators of hmpA and nnrS.
Davies et al. (22
) recently demonstrated a growth defect in a strain of V
lacking the prxA
gene, which encodes a putative peroxireductase. They used a large, short-lived bolus of NO under aerobic conditions and found that the strain exhibited a delayed log phase. In the presence of a low level of continuously released NO, a strain lacking prxA
exhibited no defect compared to the wild type. Furthermore, the expression of prxA
was not increased in the presence of NO but was dramatically increased in the presence of H2
. We suspect that PrxA is important for resistance to reactive oxygen species that may have been generated under aerobic conditions in the presence of large amounts of NO, but we conclude that it plays no role directly related to NO detoxification.
In summary, we have demonstrated the importance of the NorR regulon in NO sensing and resistance to NO toxicity. Furthermore, we identified the importance of NO detoxification genes during extended colonization of the mouse intestine. Our work highlights the role of resistance to chemical stresses in the successful survival of V. cholerae during infection and ultimately its ability to cause disease.