The Salmonella typhimurium AhpC polypeptide is not essential for virulence in BALB/c mice but is recognized as an antigen during infection

Abstract

The OxyR regulon is known to mediate protection against oxidizing agents in Salmonella typhimurium. We reported previously that ahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K. P. Francis, P. D. Taylor, C. J. Inchley, and M. P. Gallagher, J. Bacteriol. 179:4046-4048, 1997). We now report on the effects of disrupting ahp or oxyR on virulence in a BALB/c mouse model. Surprisingly, insertion of a Mudlux derivative within ahpC was found to result in attenuation, while irreversible inactivation of the locus through insertion of a cml cassette did not. An SL1344 derivative carrying an oxyR::kan disruption was also found to be as virulent as the parental strain. Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation. These results indicate that, although not essential for virulence, AhpC is expressed by S. typhimurium during infection of BALB/c mice and constitutes a target for the immune system.

FIG. 1

Disruption of the S. typhimurium ahp and oxyR loci. (A) The ahpCF locus of S. typhimurium was amplified by PCR (see Materials and Methods) and inserted into the BamHI and HindIII sites of pBR322, forming pPDT5. Subsequently, the PCR-amplified cml gene of pBR325 was inserted into the MluI and HpaI sites of the cloned ahp locus, resulting in the disruption of both ahpC and ahpF (pPDT6). The ahp locus was then liberated by PstI digestion, recircularized with DNA ligase (Intermediate), and electroporated into SL1344 in order to disrupt the corresponding chromosomal locus. Relevant restriction sites are shown. Plasmid DNA is shown as a thick line, and chromosomal DNA is shown as a thin line. C′ and ′F represent truncation of the 3′ and 5′ regions of ahpC and ahpF, respectively. (B) Southern blot of genomic DNAs from SL1344 and derivatives carrying an ahp::cml insertion. Samples of genomic DNA from SL1344 (lane 1) or from mutants that had undergone a single crossover event (lane 2) or a double crossover event (lane 3) (MPG473) are shown following digestion with HpaI. The ahpC gene, amplified by PCR from SL1344 with primers 7 and 8 (see Materials and Methods), was used as the probe. The positions of relevant DNA molecular size markers are indicated. (C) The oxyR locus of S. typhimurium was amplified by PCR (see Materials and Methods) and inserted into pGEM-T, forming pPDT7. Subsequently, a HincII fragment from pUC4-K, carrying the kanamycin resistance gene, was inserted into the SmaI site of the cloned oxyR locus, resulting in disruption (pPDT8). The oxyR locus was then liberated by EcoRI digestion, recircularized with DNA ligase (Intermediate), and electroporated into SL1344 in order to disrupt the corresponding chromosomal locus. Thick and thin lines are as in panel A. (D) Southern blot of genomic DNAs from SL1344 and a derivative carrying an oxyR::kan insertion. Samples of genomic DNA from SL1344 (lane 1) or from a mutant that had undergone a double crossover event (lane 2) (MPG484) are shown following digestion with HpaI. Probing was carried out with the oxyR gene, which was amplified by PCR from SL1344 with primers 5 and 6 (see Materials and Methods). The positions of relevant DNA molecular size markers are indicated.

FIG. 2

Measurement of CMI against AhpC during the course of infection of BALB/c mice with S. typhimurium. Groups of six female BALB/c mice (8 to 10 weeks old) were injected intraperitoneally with approximately 10⁵ CFU of attenuated S. typhimurium MPG479 in 100 μl of PBS or with 100 μl of PBS alone. The capacity for CMI was measured by subcutaneous (s.c.) injection of the RHFP with 40 μg (in 50 μl of PBS) of purified His-tagged AhpC 33 days (d) (A) or 104 days (C) postinfection or with mouse albumin 33 days postinfection (B). In each case, the LHFP was also injected with 50 μl of PBS. The thicknesses of the RHFP and LHFP were measured from three different orientations after 24 and 48 h, and the increase in the footpad thickness was expressed as a ratio [(RHFP/LHFP) × 100)] (mean ± standard error of the mean). LHFP = 100% (dashed line).

FIG. 3

BALB/C mice develop humoral immunity to AhpC during the course of infection with S. typhimurium. Western blot analysis was performed on pooled sera from groups of mice prior to infection with MPG479 (B) or at 14 (C) or 28 (D) days postinfection. (A) Standard SDS-PAGE (12.5% [vol/vol] acrylamide) loaded with a whole-cell lysate from SL1344 (lane 1) or with 2 μg of purified His-tagged AhpC (lane 2). The order of sample loading is maintained throughout the panels. Bound antibody was detected with a rabbit anti-mouse antibody conjugated to alkaline phosphatase as previously described (38). The migration positions of prestained molecular mass markers (in kilodaltons) are indicated (M).