INVESTIGADORES
GIACOMODONATO Monica Nancy
congresos y reuniones científicas
Título:
A Salmonella enteritidis dam mutant of leaky phenotype as a potential vaccine strain
Autor/es:
GIACOMODONATO MN, SARNACKI SH, SISTI F, CACCURI R, CERQUETTI MC
Lugar:
Alghero, Cerdeña, Italia
Reunión:
Conferencia; 1st ASM Conference on Salmonella: Epidemiology, Pathogenesis and Vaccine Development; 2003
Institución organizadora:
American Society for Microbiology
Resumen:
DNA adenine methylase (Dam) protein is a global regulator of bacterial gene expression. Dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. S. typhimurium strains lacking Dam protein are excellent vaccines. They are innocuous, unable to invade enterocytes or to cause cytotoxicity and they confer protective immunity. Dam mutants of S. enteritidis have been less analyzed; it was shown that S. enteritidis strains lacking Dam protein fail to generate protective immunity. We obtained a Dam mutant of S. enteritidis, named SD1, that bears a defective Dam (ten amino-acid shorter). SD1 mutant shows a leaky phenotype, its DNA has methylated and unmethylated adenines. We studied S. enteritidis SD1 as a potential vaccine strain. Mice were immunized intragastrically and challenged orally. In some experiments mice received the inocula into the ileal loop. We found that SD1 mutant is highly innocuous (oral LD50 >109 cfu). Light and electron microscopy revealed histological changes in Peyer?s patches (PP), suggesting that SD1 is able to induce cytotoxicity. Moreover, SD1 was found inside the PP 60 min after inoculation into the ileal loop. In agreement with these results SipA. SipB and SipC proteins were found in culture supernatants. Immunization with SD1 resulted in a more efficient clearance of wild type (wt) S. enteritidis from spleen, compared with control animals. In separate experiments it was found that 40% of the immunized mice survived the challenge with the wt strain. Thus, leaky mutant SD1 induces better protection than S. enteritidis strains lacking Dam. Survival rates found, however, were not as high as those reported for dam mutants of S. typhimurium. The lower efficacy of SD1 to induce protection may be related to a defective LPS. Silver-stained SDS-PAGE revealed that SD1 lacks O9 antigen, the immunodominant epitope involved in protection against S. enteritidis. Also, a defective LPS could affect the expression of IFN-g and iNOS which play a critical role in host protection against Salmonella. In fact, SD1 induced lower levels (p < 0.05) of intestinal IFN-g than wt strain (509 + 62 vs 798 + 82 pg/ mg of protein, respectively) measured by ELISA. A reduced capacity of SD1 to generate nitrites in plasma was found 48h after infection, 8.5 + 0.2 mM compared to 33,4 + 1.89 mM induced by the wt strain. Also Western blot analysis showed a delayed translocation of NFkB in the ileal loop inoculated with SD1 compared with the wt strain. In summary, our results demonstrate that S. enteritidis SD1 bearing a defective Dam invades and induces histological changes in PP. These findings, absent in mutants lacking Dam, would account for the moderate protection induced by SD1 strain. The lack of O9 antigen in SD1 mutant would explain the failure to achieve higher protection against challenge with wt S. enteritidis.