Hypermutation-induced in vivo oxidative stress resistance enhances Vibrio cholerae host adaptation

Bacterial pathogens are highly adaptable organisms, a quality that enables them to overcome changing hostile environments. For example, Vibrio cholerae, the causative agent of cholera, is able to colonize host small intestines and combat host-produced reactive oxygen species (ROS) during infection. To dissect the molecular mechanisms utilized by V. cholerae to overcome ROS in vivo, we performed a whole-genome transposon sequencing analysis (Tn-seq) by comparing gene requirements for colonization using adult mice with and without the treatment of the antioxidant, N-acetyl cysteine. We found that mutants of the methyl-directed mismatch repair (MMR) system, such as MutS, displayed significant colonization advantages in untreated, ROS-rich mice, but not in NAC-treated mice. Further analyses suggest that the accumulation of both catalase-overproducing mutants and rugose colony variants in NAC- mice was the leading cause of mutS mutant enrichment caused by oxidative stress during infection. We also found that rugose variants could revert back to smooth colonies upon aerobic, in vitro culture. Additionally, the mutation rate of wildtype colonized in NAC- mice was significantly higher than that in NAC+ mice. Taken together, these findings support a paradigm in which V. cholerae employs a temporal adaptive strategy to battle ROS during infection, resulting in enriched phenotypes. Moreover, ΔmutS passage and complementation can be used to model hypermuation in diverse pathogens to identify novel stress resistance mechanisms.


Fig. B.
Chromosomal complementation of mutS. Cultures of wildtype, ∆mutS, and chromosomally inserted (in lacZ locus) mutS in ∆mutS were grown in LB until saturated and then plated on LB agar and LB agar + 50 µg/ml rifampicin. After overnight growth at 37˚C, rifampicin resistant colonies were scored. ****: One-way ANOVA P < 0.0001. ns: no significance.

Fig. C.
The effect of mutS on V. cholerae growth. Wildtype and ∆mutS growth in LB (shaking)(A) and AKI medium (standing)(B). OD600 was measured. C. Growth of WT* and ∆mutS* in LB and AKI to mid-log phase. OD600 was measured and compared with their parental strains.

A.
B.

Fig. D.
Expression of catalase genes in ΔmutS* isolates. Mid-log cultures of wildtype, ΔmutS, and selected ΔmutS * were induced with 500 µM H2O2 for 1 hr. Total RNA was extracted and cDNA was synthesized. Reverse transcription-quantitative PCR (qRT-PCR) was carried out and normalized against 16S rRNA as the internal standard. Error bars represent means and SDs from three independent assays. *: One-way ANOVA P <0.05 (compared to wildtype).

Fig. E.
Colonization of ROS-sensitive mutants in NACmice. A. ΔkatGB. ∆mutS or ∆mutS ∆katGkatB mutants were mixed with wildtype at 1:1 ratio and intragastrically administered to NACmice. Fecal pellets were collected from each mouse at 4-day PI and plated onto X-gal plates with appropriate antibiotics. The competitive index (CI) was calculated as the ratio of mutants to wildtype normalized to the input ratio. Horizontal line: mean CI of 5 mice. **: Mann-Whiteney test P value < 0.01. B. ΔoxyR. ∆oxyR mutants were mixed with wildtype in a 1:1 ratio and intragastrically administered to NACmice. Fecal pellets were collected from each mouse at 4-day PI and plated onto X-gal plates with 10 µg/ml catalase and appropriate antibiotics. The competitive index (CI) was calculated as the ratio of mutants to wildtype normalized to the input ratio. Horizontal line: mean CI of 5 mice. **: Mann-Whiteney test P value < 0.01.

A. B.
10 A. pBB1 expression in ∆mutS*. Wildtype, ∆mutS, and ∆mutS* rugose variants containing a HapR-regulated luxCDABE (pBB1) [68] were grown in LB with appropriate antibiotics at 30ºC overnight, diluted to a concentration of 1:100 in fresh LB and transferred to white opaque 96 well plates and incubated while shaking at 30ºC. Luminescence was read at OD600 = 1. B.
Colonization. Wildtype and ∆hapR were co-inoculated into 6-week-old CD-1 mice with or without NAC treatment. Fecal pellets were collected after 5 days and plated onto selective plates. The competitive index was calculated as the ratio of mutant to wildtype colonies normalized to the input ratio.

B.
A.  were mixed in a 1:1 ratio and approximately 10 5 cells were intragastrically inoculated into 5-dayold CD-1 suckling mice. After a 20-hr period of incubation, mice were sacrificed. Small intestines were harvested and homogenized, the ratio of mutants to WT bacteria was determined by plating onto LB agar containing antibiotics and X-Gal. B.&C. Overnight cultures of wildtype, ∆mutS and ∆mutS* containing PtcpA-luxCDABE transcriptional fusion plasmids were inoculated 1:10000 into AKI medium [37] and incubated without shaking at 37°C for 4 hrs, followed by shaking at 37°C for an additional 3 hrs. Luminescence was then measured at the indicated time points and normalized to OD600 (B). At the final time point, 10 9 cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using anti-TcpA antiserum (C).