A Temperature-Sensitive dam Mutant of Salmonella enteritidis as an Experimental Vaccine Candidate.

GIACOMODONATO MN, GOREN MB, SARNACKI SH, SORDELLI DO, CERQUETTI MC.

Washington DC, USA

Congreso; 103 ed General Meeting of American Society for Microbiology; 2003

American Society for Microbiology

DNA adenine methylase (Dam) acts as a global regulator of gene expression affecting a wide range of cellular functions. Salmonella spp. dam mutants have been recently presented as promising vaccine candidates. Previously, we obtained and characterized insertional mutants of Salmonella spp. with a temperature-sensitive Dam protein. Here we study the ability of one of these mutants , S. enteritidis mutant SD1, to interact with the intestinal mucosa and to elicit a protective immune response in a murine model for salmonellosis. Eight-week old Balb/c mice were used in all the experiments. Animals were inoculated intraperitoneally (ip) and intragastricaly (ig) with different doses of mutant SD1. Results show that SD1 mutant is highly innocuous as indicated by LD50s ( > 105 CFU and > 109 CFU for the ip and ig routes respectively). Unlike other dam mutants of Salmonella spp. investigated, SD1 was able to reach internal organs as early as 20 hs after ig administration of 109 CFU. Forty-eight hs post inoculation the attenuated mutant was recovered from Peyer´s patches (median: 2.1 x 104 CFU ; range: 2.0 x 103 -2.4 x 104)and spleen (median: 1.4 x 106 CFU ; range: 4.6 x 103-1.1 x107 ). SD1 mutant elicited protective immunity in mice. Sixty percent of the animals receiving 3 x 108 UFC ig of the attenuated strain were protected against challenge with the virulent strain of S. enteritidis. Short term experiments were also performed to determine whether SD1 was able to activate NFkB and to induce IFN-gamma secretion in the intestinal mucosa. Western blot analyses revealed that SD1 mutant induces degradation of IkB-alpha within 60 min upon bacterial inoculation. In addition, it was found that SD1 mutant is able to induce significant levels (p < 0.05) of IFN-gamma in the gut early after infection (660 +/- 91 pg/ug protein) compared with control mice (350 +/- 75 pg/ug protein). Altogether our results showed that the modified Dam protein bore by SD1 mutant of S. enteritidis attenuates its virulence without affecting the interaction with the intestinal epithelium. Based on these results SD1 mutant warrants further evaluation as a candidate S. enteritidis vaccine.