O-antigen chain-length distribution in Salmonella enterica serovar Enteritidis is regulated by oxygen availability.

Lipopolysaccharide (LPS) consists of three covalently linked domains: the lipid A, the core region and the O antigen (OAg), consisting of repeats of an oligosaccharide. Salmonella enterica serovar Enteritidis (S. Enteritidis) produces a LPS with two OAg preferred chain lengths: a long (L)-OAg controlled by WzzSE and a very long (VL)-OAg controlled by WzzfepE. In this work, we show that OAg produced by S. Enteritidis grown in E minimal medium also presented two preferred chain-lengths. However, a simultaneous and opposing change in the production of L-OAg and VL-OAg was observed in response to oxygen availability. Biochemical and genetics analyses indicate that this process is regulated by transcriptional factors Fnr and ArcA by means of controlling the transcription of genes encoding WzzSE and WzzfepE in response to oxygen availability. Thus, our results revealed a sophisticated regulatory mechanism involved in the adaptation of S. Enteritidis to one of the main environmental cues faced by this pathogen during infection.

A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines.

BACKGROUND: Generation of tolerogenic dendritic cells (TolDCs) for therapy is challenging due to its implications for the design of protocols suitable for clinical applications, which means not only using safe products, but also working at defining specific biomarkers for TolDCs identification, developing shorter DCs differentiation methods and obtaining TolDCs with a stable phenotype. We describe here, a short-term protocol for TolDCs generation, which are characterized in terms of phenotypic markers, cytokines secretion profile, CD4+ T cell-stimulatory ability and migratory capacity. METHODS: TolDCs from healthy donors were generated by modulation with dexamethasone plus monophosphoryl lipid A (MPLA-tDCs). We performed an analysis of MPLA-tDCs in terms of yield, viability, morphology, phenotypic markers, cytokines secretion profile, stability, allogeneic and antigen-specific CD4+ T-cell stimulatory ability and migration capacity. RESULTS: After a 5-day culture, MPLA-tDCs displayed reduced expression of costimulatory and maturation molecules together to an anti-inflammatory cytokines secretion profile, being able to maintain these tolerogenic features even after the engagement of CD40 by its cognate ligand. In addition, MPLA-tDCs exhibited reduced capabilities to stimulate allogeneic and antigen-specific CD4+ T cell proliferation, and induced an anti-inflammatory cytokine secretion pattern. Among potential tolerogenic markers studied, only TLR-2 was highly expressed in MPLA-tDCs when compared to mature and immature DCs. Remarkable, like mature DCs, MPLA-tDCs displayed a high CCR7 and CXCR4 expression, both chemokine receptors involved in migration to secondary lymphoid organs, and even more, in an in vitro assay they exhibited a high migration response towards CCL19 and CXCL12. CONCLUSION: We describe a short-term protocol for TolDC generation, which confers them a stable phenotype and migratory capacity to lymphoid chemokines, essential features for TolDCs to be used as therapeutics for autoimmunity and prevention of graft rejection.

Spontaneous excision of the Salmonella enterica serovar Enteritidis-specific defective prophage-like element phiSE14.

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an approximately 12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5' end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of phiSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of phiSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of phiSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that phiSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of phiSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications.