LcrV synthesis is altered by DNA adenine methylase overproduction in Yersinia pseudotuberculosis and is required to confer immunity in vaccinated hosts

Abstract

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (DamOP Yersinia) are attenuated, confer robust protective immune responses, and synthesize or secrete several Yersinia outer proteins (Yops) under conditions that are nonpermissive for synthesis and secretion in wild-type strains. To understand the molecular basis of immunity elicited by DamOP Yersinia, we investigated the effects of Dam overproduction on the synthesis and localization of a principal Yersinia immunogen, LcrV, a low-calcium-responsive virulence factor involved in Yop synthesis, localization, and suppression of host inflammatory activities. Dam overproduction relaxed the stringent temperature and calcium regulation of LcrV synthesis. Moreover, the LcrV-dependent synthesis and localization of the actin cytotoxin, YopE, were shown to be relaxed in DamOP cells, suggesting that the synthesis and localization of Yops can occur via both LcrV-dependent and -independent mechanisms. Last, the immunity conferred by DamOP Yersinia was strictly dependent on the presence of LcrV, which may result from its role (i) as an immunogen, (ii) as an immunomodulator of host anti-inflammatory activities, or (iii) in the altered synthesis and localization of Yops that could contribute to immunogen repertoire expansion.

FIG. 1.

The high-temperature and low-calcium dependence of LcrV synthesis is relaxed in Dam-overproducing Y. pseudotuberculosis. Whole-cell (WC), membrane (Memb), and supernatant (Sup) fractions (12) were prepared from wild-type (WT) and Dam-overproducing (OP) Y. pseudotuberculosis grown under the indicated conditions according to methods described previously (12, 44, 45). For each growth condition, total protein extracts corresponding to 2.0 × 10⁶ cells (∼20 μg of protein/well) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Pierce), and probed with mouse anti-LcrV monoclonal antibodies (1:4,000 dilution). Peroxidase-conjugated sheep anti-mouse immunoglobulin G (1:40,000 dilution; Amersham Biosciences), was used as the secondary antibody, and hybridization was detected by chemiluminescence using Supersignal West Femto Maximum Sensitivity Substrate (Pierce) followed by a 2-min exposure to film. Inspection of corresponding Coomassie-stained gels showed similar band intensities of nonregulated proteins under all conditions tested (data not shown). Western analysis of lcrV⁺ and lcrV⁻ strains confirmed that the 37-kDa protein was LcrV (data not shown).

FIG. 2.

The LcrV dependence of YopE synthesis and localization is relaxed under Dam-overproducing conditions in Y. pseudotuberculosis. Whole-cell (WC), membrane (Memb), and supernatant (Sup) fractions (12) were prepared from dam wild-type (WT) and Dam-overproducing (OP) Y. pseudotuberculosis containing (A) or lacking (B) the lcrV gene. For each growth condition (12, 44, 45), total protein extracts corresponding to 2.0 × 10⁶ cells (∼20 μg of protein/well) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Pierce), and probed with rabbit anti-YopE polyclonal antibodies (1:50,000 dilution). Peroxidase-conjugated donkey anti-rabbit immunoglobulin G was used as the secondary antibody (1:20,000 dilution; Amersham Biosciences), and hybridization was detected by chemiluminescence using Supersignal West Femto Maximum Sensitivity Substrate (Pierce) followed by a 30-s (A) or 2-min (B) exposure to film. Inspection of corresponding Coomassie-stained gels showed similar band intensities of nonregulated proteins under all conditions tested (data not shown). Western analysis of yopE⁺ and yopE⁻ strains confirmed that the 23-kDa protein was YopE (23).