Host Response to a dam Mutant of Salmonella enterica Serovar Enteritidis with a Temperature-Sensitive Phenotype

Abstract

The temperature-sensitive dam mutant strain of Salmonella enterica serovar Enteritidis SD1 is highly attenuated and induces innate and protective immunity in mice. SD1 activates NF-B and induces gamma interferon secretion. Early interaction of the SD1 mutant with intestinal epithelial cells was associated with ruffling of enterocytes. Invading bacteria were found inside Peyer's patches after inoculation.Salmonella species dam mutants are highly attenuated for virulence and have been proposed as live vaccines (10, 11). The safety of dam mutants of Salmonella spp. is enhanced by the inability of the microorganisms to invade enterocytes or to be toxic to M cells of ileal Peyer's patches (7). These features, however, together with the fact that dam mutants present a defective induction of inducible nitric oxide (NO) synthase and gamma interferon (IFN-␥) (22), would limit their use as bacterial carriers or delivery systems.In the last few decades, Salmonella enterica serovar Enteritidis has emerged as a major cause of food-borne illness worldwide; in Argentina, for instance, the proportion of salmonellosis cases attributed to this pathogen showed a 275-fold increase in that time (12,15,18). In contrast, few studies using Salmonella serovar Enteritidis dam mutants as vaccine strains have been published. Earlier, we obtained a dam insertion mutant of Salmonella serovar Enteritidis named SD1. The insertion dam-231::Tn10dTet rendered in the SD1 mutant a functional (but defective) Dam that was 10 amino acids shorter than the native protein (3; M. N. Giacomodonato, S. H. Sarnacki, F. Sisti, R. Caccuri, and M. C. Cerquetti, Am. Soc. Microbiol. Conf. Salmonella: pathogenesis, epidemiology, and vaccine development, abstr. 106(A) p. 69, 2003). Some differences were found between the null dam mutant TT11694 of Salmonella serovar Typhimurium and the SD1 strain (Table 1). Filamentation and sensitivity to 2-aminopurine were observed in the SD1 mutant only at 37°C, whereas the TT11694 strain filamented and was sensitive to 2-aminopurine regardless of the incubation temperature. Here, we investigated the ability of the temperature-sensitive dam mutant of Salmonella serovar Enteritidis SD1 to interact, in vivo, with the intestinal mucosa and to induce protective immunity in mice.The SD1 mutant induces early host responses in the murine model. To determine whether the SD1 mutant was able to induce early responses in murine intestines, short-term experiments using an ileal loop (13) were performed. Infection with the wild-type strain of Salmonella serovar Enteritidis resulted in the rapid but transient degradation of IB-␣. Similarly, SD1 inoculation resulted in IB-␣ degradation, although it was slower degradation than that seen with the wild-type strain (Fig. 1). The cytokines secreted 60 min after bacterial inoculation were determined by enzyme-linked immunosorbent assay. The SD1 mutant was able to induce significantly higher levels (P Ͻ 0.05) of IFN-␥ in the gut early after inoculation (660 Ϯ 101 pg/g of protein) than levels induced ...

Epigenetic Gene Regulation in the Bacterial World

“…In Salmonella, Haemophilus, and certain strains of Yersinia pseudotuberculosis, lack of Dam methylation causes attenuation of virulence in model animals (89,93,107,201,252,274). In other pathogens, virulence attenuation is observed if Dam methylase is overproduced (56,136).…”

Regulation of the Salmonella enterica std Fimbrial Operon by DNA Adenine Methylation, SeqA, and HdfR

“…dam mutants of Salmonella enterica are severely attenuated in the mouse model: lack of DNA adenine methylation causes a 10,000 increase in the oral 50% lethal dose , and a 1,000-fold increase in the intraperitoneal 50% lethal dose (17,19,21). This extreme attenuation reflects the pleiotropy of dam mutations, which cause reduced invasion of epithelial cells (17), reduced cytotoxicity after infection of M cells (17), inefficient colonization of Peyer's patches and mesenteric lymph nodes (17,21), sensitivity to bile (22,40,41), envelope instability accompanied by leakage of proteins (41), reduced motility (3), and probably additional defects still to be discovered.…”

Dam Methylation Controls O-Antigen Chain Length in Salmonella enterica Serovar Enteritidis by Regulating the Expression of Wzz Protein

“…We observed previously that Salmonella serovar Enteritidis mutant SD1 expressing a truncated Dam protein (15) does not agglutinate with anti-O9 serum (14). To better understand the role of Dam in LPS expression, we constructed a dam deletion mutant of Salmonella serovar Enteritidis strain #5694 (SE⌬dam) (5).…”

“…Recently, dam mutants of S. enterica serovar Typhimurium have been analyzed as potential live vaccines to prevent salmonellosis in birds and cattle (10,11). We have reported previously that an S. enterica serovar Enteritidis dam mutant expressing a truncated Dam protein is attenuated (15). This mutant has limited protective capacity as a live vaccine (55) and is unable to agglutinate in the presence of specific antibodies against O9 polysaccharide (14,55), suggesting that there is a defective LPS.…”