Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide.

Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.[1] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26 kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.

Comparative roles of clpA and clpB in the survival of S. Typhimurium under stress and virulence in poultry.

By assisting in the proteolysis, disaggregation and refolding of the aggregated proteins, Caseinolytic proteases (Clps) enhance the cellular survival under stress conditions. In the current study, comparative roles of two such Clps, ClpA (involved in proteolysis) and ClpB (involved in protein disaggregation and refolding) in the survival of Salmonella Typhimurium (S. Typhimurium) under different stresses and in virulence have been investigated. clpA and clpB gene deletion mutant strains (∆clpA and ∆clpB) of S. Typhimurium have been hypersensitive to 42 °C, HOCl and paraquat. However, the ∆clpB strain was comparatively much more susceptible (p < 0.001) to the above stresses than ∆clpA strain. ∆clpB strain also showed reduced survival (p < 0.001) in poultry macrophages. The hypersusceptibilities of ∆clpB strain to oxidants and macrophages were restored in plasmid based complemented (∆clpB + pclpB) strain. Further, the ∆clpB strain was defective for colonization in the poultry caecum and showed decreased dissemination to the spleen and liver. Our findings suggest that the role of ClpB is more important than the role of ClpA for the survival of S. Typhimurium under stress and colonization in chickens.

Role of different receptors and actin filaments on Salmonella Typhimurium invasion in chicken macrophages.

Bacterial attachment to host cell is the first event for pathogen entry. The attachment is mediated through membrane expressed adhesins present on the organism and receptors on the cell surface of host. The objective of this study was to investigate the significance of Fc receptors (FcRs), actin filament polymerization, mannose receptors (MRs), carbohydrate moieties like N-linked glycans and sialic acid on chicken macrophages for invasion of S. Typhimurium. Opsonisation of S. Typhimurium resulted in three folds more invasion in chicken monocyte derived macrophages. Cytochalasin D, an inhibitor of actin filament polymerization prevented uptake of S. Typhimurium. Pre-incubation of macrophages with cytochalasin D, showed severe decrease (28 folds) in S. Typhimurium invasion. Next we attempted to analyse the role of carbohydrate receptors of macrophages in S. Typhimurium invasion. Treatment of macrophages with methyl α-d-mannopyranoside, PNGase F and neuraminidase, showed 2.5, 5 and 2.5 folds decrease in invasion respectively. Our data suggest that deglycosylation of N-linked glycans including sialic acid by PNGase F is more effective in inhibition of S. Typhimurium invasion than neuraminidase which removes only sialic acid. These findings suggested FcRs, actin filament polymerization, MRs, N-linked glycans and sialic acid may act as gateway for entry of S. Typhimurium.