Dexamethasone modulates Salmonella enterica serovar Typhimurium infection in vivo independently of the glucocorticoid-inducible protein annexin-A1.

Salmonella enterica serovar Typhimurium (S. Typhimurium) infection causes an inflammatory response through activation of Toll-like receptor 4 by lipopolysaccharide. Dexamethasone, a glucocorticoid analogue, suppresses inflammatory responses by many mechanisms including inhibition of the lipopolysaccharide-induced production of proinflammatory mediators. There is little information on the effect of glucocorticoids on murine salmonellosis. In this study, we treated susceptible BALB/c mice by subcutaneous implantation of slow-release dexamethasone pellets before infection with S. Typhimurium. Dexamethasone promotes bacterial growth early in infection and induces a dose-dependent increase in bacterial growth within mouse livers and spleens. The bacterial load in organs from infected placebo-treated mice was lower than that in dexamethasone-treated mice. Glucocorticoids inhibit lipopolysaccharide-induced inflammation partially through the steroid-inducible protein annexin-A1 (ANXA1). Infection of wild-type and ANXA1 knock-out mice with S. Typhimurium led to similar organ bacterial loads. ANXA1 also did not affect the bacterial load in organs from infected dexamethasone-treated mice. This suggests that glucocorticoids, independently of ANXA1, accelerate S. Typhimurium growth in vivo in susceptible BALB/c mice

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria

Comprehensive Identification of Salmonella enterica Serovar Typhimurium Genes Required for Infection of BALB/c Mice

Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH), has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input) with equivalent data produced after passage of the library through mice (output) enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines