Signature-Tagged Mutagenesis to Identify Virulence Genes in Salmonella choleraesuis

Signature-tagged mutagenesis is a functional genomics approach to identify bacterial virulence genes by simultaneously screening multiple mutants in a single host animal. Avirulent (attenuated) mutants are identified by negative selection (failure to colonize the host). The method was recently developed to investigate Salmonella typhimurium in a mouse model of human typhoid fever. We modified the protocol to investigate virulence genes of S. choleraesuis in its natural host, the pig. First, we generated random, knock-out (null) mutations in S. choleraesuis using transposon-mediated insertion of unique, signature-tagged (40 bp), kanamycin resistance cassettes. To validate the modified protocol, a test pool of 45 mutants was inoculated orally or intraperitoneally (systemic infection) into pigs. Three of the mutants were not identified from cultures of the mesenteric lymph nodes obtained four days after infection. Attenuation of these three candidate attenuated mutants was confirmed by mixed challenge growth experiments (growth of a 1:1 mixture of mutant and wild type S. choleraesuis) in broth cultures and in pigs. All three mutants were attenuated for infection of pigs as the mutants were at a marked disadvantage relative to the wild type bacteria for growth. For one mutant, the competitive indices (ratios of mutant to wild type bacteria) were 0.15 and 0.03 for bacteria in the intestines and mesenteric lymph nodes, respectively; for the other two mutants, the indices were about 0.001 in either tissue. In contrast, none of the mutants were at a growth disadvantage in broth cultures. As an additional control, two random mutants recovered from the pigs inoculated with the pool (and therefore not candidates for attenuation) were also tested in the mixed challenge experiments. As expected, the competitive growths of these mutants in culture medium and in pigs were similar to that of the wild type. In one of the three attenuated mutants the inactivated gene has been identified, after cloning and sequencing, as hilA . In mouse models of S. typhimurium infection, hilA is associated with enteric invasion, but not systemic proliferation. Because the hilA mutant failed to colonize pigs irrespective of oral or systemic inoculation, its function in pigs is likely to be more complex than in the mouse. Our data show signature-tagged mutagenesis can be used to identify Salmonella genes associated with virulence in pigs. As we continue screening mutants, we expect to identify novel genes related to pathogenicity and we are expanding our studies to include S. typhimurium.