TABLE 3.
Dam overproduction confers a virulence defect in Y. pseudotuberculosis and V. cholerae
| Strain | Relevant genotypea | Oral LD50 ratiob (mutant/wild type) | Competitive indexc |
|---|---|---|---|
| MT2294 | DamOPY. pseudotuberculosis | >6,000 | <10−4 |
| MT2284 | DamOPV. cholerae | ND | 0.218 |
Bacterial strains are derivatives of Y. pseudotuberculosis strain YPIIIpYV and V. cholerae strain O395. Dam-overproducing (DamOP) strains of Y. pseudotuberculosis (MT2294) and V. cholerae (MT2284) contain E. coli dam on chloramphenicol- and tetracycline-resistant derivatives of the high-copy-number recombinant plasmid pTP166 (29) and the medium-copy-number plasmid pWKS30 (43), respectively, in Dam mutant (Δdam::Kn) genetic backgrounds. (Dam overproduction from the high-copy-number plasmid pTP166-Cm in V. cholerae was deleterious to V. cholerae cell growth as evidenced by a 50% decrease in the rate of growth on rich medium.) Since dam is essential for viability in Y. pseudotuberculosis and V. cholerae, the loss of the Dam-overproducing plasmids in dam mutant backgrounds is lethal for both pathogens.
The oral LD50 ratio (the LD50 of the Dam-overproducing strain divided by the LD50 of wild-type bacteria) was determined by infecting 18 BALB/c mice with 1.56 × 1011 cells of the Y. pseudotuberculosis Dam-overproducing strain MT2294 as described previously (20); 18 of 18 mice survived this challenge dose, and no visible signs of infection were observed. The peroral LD50 of wild-type Y. pseudotuberculosis (2.5 × 107) was determined by Monack et al. (30). ND, not determined.
For Y. pseudotuberculosis infection, six BALB/c mice were gastrointubated with 8.0 × 108 Yersinia dam mutant cells containing the Dam-overproducing plasmid from strain MT2294 and 8.0 × 108 cells of the wild type. After 7 days, mice were sacrificed, spleens were recovered and homogenized, and bacteria were enumerated by direct colony counting as described previously (5). Of the 106 to 107 Yersinia organisms recovered from each of six spleens, none contained the Dam-overproducing plasmid. For V. cholerae infection, six CD-1 mice were gastrointubated with a 1:1 ratio of Dam-overproducing cells (MT2284) to wild-type cells; 24 h postinfection, mice were sacrificed and bacterial numbers were determined from the intestine as described previously (7). The attenuation conferred was significant according to the two-tailed Fisher exact test (P < 0.05).