Immunosuppression induced by Salmonella involves inhibition of tyrosine phosphorylation in murine T lymphocytes

Abstract It is well known that facultative intracellular pathogens such as Salmonella suppress the host immune system. In the present study we attempted to clarify the mechanism responsible for the suppression of T-cell proliferation in mice infected with Salmonella typhimurium . The proliferation of murine spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were infected with S. typhimurium , but not with Eschirichia coli . The suppression of T-cell proliferation did not necessarily parallel the level of interleukin-2 (IL-2) secretion, and was not restored by treatment with a calcium ionophore, indomethacin or IL-2. Only phorbol 12-myristate-13 acetate (PMA), an activator of protein kinase C (PKC), caused a slight recovery of cell proliferation with an augmentation of IL-2 secretion. Furthermore, Western blotting using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 68- and 57-kDa proteins in murine splenic T-cells was inhibited by S. typhimurium infection. Also, the inhibition of tyrosine phosphorylation was not restored by treatment with PMA. These results suggest that the suppression of T-cell proliferation induced by Salmonella infection may be regulated by inhibition of tyrosine phosphorylation in T-cells, although the inhibition is not associated with PKC activation and subsequent IL-2 secretion of T cells.     

Proteomic Analysis of Salmonella-modified Membranes Reveals Adaptations to Macrophage Hosts

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Highlights

• •Affinity-based proteomics of infected macrophage cells.
• •Salmonella-modified membranes exhibit host-specific composition.
• •Proteome differences explain some host-dependent pathophysiological differences.

     

Membrane localisation of a UDP-sugar hydrolase, in Salmonella, is by an uncleaved N-terminal signal peptide

Abstract Most isolates of Salmonella contain two unrelated UDP-sugar hydrolases, one of which, encoded by the ushB gene, is inner membrane-associated. Previous studies showed that this enzyme contains a typical N-terminal signal peptide; the evidence also indicated, however, that this peptide is not cleaved, and serves to anchor the UshB protein in the inner membrane. In this report, we present strong evidence that this is indeed the case by using ushB'-'blaM fusions to demonstrate that this signal peptide is capable of localising β-lactamase to the inner membrane. We also present evidence that UshB is located on the exterior (periplasmic) side of the membrane, and hence has an 'N-terminus inside/C-terminus outside' membrane orientation, consistent with a role in the degradation of external substrates.     

Inactivation of Salmonella serovars by Pseudomonas chlororaphis and Pseudomonas fluorescens strains on tomatoes

Salmonella enterica and its serovars have been associated with pathogen contamination of tomatoes with numerous outbreaks of salmonellosis. To improve food safety, pathogen control is of immediate concern. The aim of this research was to assess the populations of natural microflora (aerobic mesophilic bacteria, lactic acid bacteria, yeasts and moulds and Pseudomonas species) on tomatoes, and evaluate the efficacy of Pseudomonas fluorescens (Pf) and Pseudomonas chlororaphis (Pc) for inactivation of Salmonella on tomatoes. Microflora were determined on sanitised and unsanitised produce and enumerated on Plate Count Agar, de Man, Rogosa and Sharpe medium, Potato Dextrose Agar and Pseudomonas Agar F media. The efficacy of Pc and Pf for inactivation of S. enterica serovars Montevideo, Typhimurium and Poona was determined on spot-inoculated tomato stem scars. The effects of storage time on bacterial populations were also investigated. On unsanitised tomatoes, lactic acid bacteria, Pseudomonas sp., aerobic mesophilic bacteria and yeasts and moulds ranged from 3.31–4.84, 3.93–4.77, 4.09–4.80 and 3.83–4.67 log CFU/g of produce, respectively. The microflora were similar at 0 and 24 storage hours on sanitised produce. The suppression of Salmonella Montevideo by P. chlororaphis and P. fluorescens on tomatoes ranged from 0.51 to 2.00 log CFU/g of produce. On Salmonella Montevideo and S. Typhimurium, the suppressive effects ranged from 0.51 to 0.95 and 0.46 to 2.00 log CFU/g of produce, respectively. The pathogen suppressive effects may be attributed to competition ability of Pseudomonas relative to Salmonella strains. Pseudomonas strains may be effective against Salmonella strains as a post-harvest application, but strain synergy is required to optimise pathogen reductions.     

Electron spin-labelling of the EutC subunit in B12-dependent ethanolamine ammonia-lyase reveals dynamics and a two-state conformational equilibrium in the N-terminal,signal-sequence-associated domain

Abstract

The B₁₂ (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine utilisation (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Toward elucidation of the molecular basis of EAL catalysis, and its intracellular trafficking and targeting to the Eut biomicrocompartment (BMC), we have applied electron spin-labelling and electron paramagnetic resonance spectroscopy to wild-type (wt) EAL from Salmonella typhimurium, by using the sulphydryl-specific, 4-maleimido-TEMPO (4MT) spin label. One cysteine residue per active site displays exceptional reactivity with 4MT. This site is identified as βC37 on the EutC subunit, by using 4MT-labeling of site-specific cysteine-to-alanine mutants, enzyme kinetics, and accessible surface area calculations. Electron paramagnetic resonance (EPR) spectra of 4MT-labelled wt EAL are collected over 200–265 K in frozen, polycrystalline water-only, and 1% v/v DMSO solvents. EPR simulations reveal two mobility components for each condition. Detectable spin probe reorientational motion of the two components occurs at 215 and 225 K with 1% v/v DMSO, relative to the water-only condition, consistent with formation of an aqueous-DMSO solvent mesodomain around EAL. Parallel trends in fast- and slow-reorientational correlation times and interconversion of the two populations with increasing temperature, indicate 4MT labelling of a single site (βC37). A two-state model is proposed, in which the fast and slow motional populations represent EAL-bound and free conformations of the EutC N-terminal domain. The approximately equal proportion of each state may represent a balance between EutC and EAL protein stability and efficient targeting to the BMC.     

Secretory autophagy of lysozyme in Paneth cells

Secretion of antimicrobial proteins is an important host defense mechanism against bacteria, yet how secretory cells maintain function during bacterial invasion has been unclear. We discovered that Paneth cells, specialized secretory cells in the small intestine, react to bacterial invasion by rerouting a critical secreted antibacterial protein through a macroautophagy/autophagy-based secretion system termed secretory autophagy. Mice harboring a mutation in an essential autophagy gene, a mutation which is common in Crohn disease patients, cannot reroute their antimicrobial cargo during bacterial invasion and thus have compromised innate immunity. We showed that this alternative secretion system is triggered by both a cell-intrinsic mechanism, involving the ER stress response, and a cell-extrinsic mechanism, involving subepithelial innate immune cells. Our findings uncover a new role for secretory autophagy in host defense and suggest how a mutation in an autophagy gene can predispose individuals to Crohn disease.     

Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein

With the development of de novo binders for protein targets from non‐related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single‐chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking “disembodied” amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein‐antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen‐based detection agents for typhoid diagnostics.