We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuated Salmonella vaccine (RASV) strains to enhance immune responses by reducing the adverse effects of high-level antigen synthesis. This system includes a chromosomal repressor gene, lacI, expressed from the arabinoseregulated araC P BAD promoter. LacI serves to regulate expression from a plasmid promoter, P trc , that directs antigen synthesis. In the presence of arabinose LacI is produced, which binds to P trc , blocking antigen synthesis. In vivo, an arabinose-poor environment, the concentration of LacI decreases with each cell division, allowing increased antigen synthesis. To optimize the system and for comparison, we altered the lacI ribosomebinding site, start codon, and/or codon content to construct RDAS strains 9095, 9959, and 9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain 9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initial in vitro assessment and the Streptococcus pneumoniae PspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression of lacI did not interfere with the capacity to induce an immune response. Strain 9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain 9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain 9241 also induced significantly greater protective efficacy against challenge with virulent S. pneumoniae. These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.

Leucine-responsive regulatory protein (Lrp) is a global gene regulator that influences expression of a large number of genes including virulence-related genes in Escherichia coli and Salmonella. No systematic studies examining the regulation of virulence genes by Lrp have been reported in Salmonella. We report here that constitutive expression of Lrp [lrp(Con)] dramatically attenuates Salmonella virulence while an lrp deletion (⌬lrp) mutation enhances virulence. The lrp(Con) mutant caused pleiotropic effects that include defects in invasion, cytotoxicity, and colonization, whereas the ⌬lrp mutant was more proficient at these activities than the wild-type strain. We present evidence that Lrp represses transcription of key virulence regulator geneshilA, invF, and ssrA-in Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2), by binding directly to their promoter regions, P hilA , P invF , and P ssrA . In addition, Western blot analysis showed that the expression of the SPI-1 effector SipA was reduced in the lrp(Con) mutant and enhanced in the ⌬lrp mutant. Computational analysis revealed putative Lrp-binding consensus DNA motifs located in P hilA , P invF , and P ssrA . These results suggest that Lrp binds to the consensus motifs and modulates expression of the linked genes. The presence of leucine enhanced Lrp binding to P invF in vitro and the addition of leucine to growth medium decreased the level of invF transcription. However, leucine had no effect on expression of hilA and ssrA or on cellular levels of Lrp. In addition, Lrp appears to be an antivirulence gene, since the deletion mutant showed enhanced cell invasion, cytotoxicity, and hypervirulence in BALB/c mice.

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrpC) mutant strains. In this work, we used chromosomal PfimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to PfimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in PfimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to PfimA without competitive exclusion. In addition, both Lrp and FimZ binding to PfimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.

Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo. We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 105 CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 109 CFU of wild-type S. Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.