Polyamines are required for virulence in Salmonella enterica serovar Typhimurium

Sensing and responding to environmental cues is a fundamental characteristic of bacterial physiology and virulence. Here we identify polyamines as novel environmental signals essential for virulence of Salmonella enterica serovar Typhimurium, a major intracellular pathogen and a model organism for studying typhoid fever. Central to its virulence are two major virulence loci Salmonella Pathogenicity Island 1 and 2 (SPI1 and SPI2). SPI1 promotes invasion of epithelial cells, whereas SPI2 enables S. Typhimurium to survive and proliferate within specialized compartments inside host cells. In this study, we show that an S. Typhimurium polyamine mutant is defective for invasion, intracellular survival, killing of the nematode Caenorhabditis elegans and systemic infection of the mouse model of typhoid fever. Virulence of the mutant could be restored by genetic complementation, and invasion and intracellular survival could, as well, be complemented by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection. Interestingly, intracellular survival of the polyamine mutant was significantly enhanced above the wild type level by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection, indicating that these polyamines function as an environmental signal that primes S. Typhimurium for intracellular survival. Accordingly, experiments addressed at elucidating the roles of these polyamines in infection revealed that expression of genes from both of the major virulence loci SPI1 and SPI2 responded to exogenous polyamines and was reduced in the polyamine mutant. Together our data demonstrate that putrescine and spermidine play a critical role in controlling virulence in S. Typhimurium most likely through stimulation of expression of essential virulence loci. Moreover, our data implicate these polyamines as key signals in S. Typhimurium virulence.     

Bile-induced curing of the virulence plasmid in Salmonella enterica serovar Typhimurium

Exposure to bile induces curing of the virulence plasmid in Salmonella enterica serovar Typhimurium (pSLT). Disruption of the ccdB gene increases pSLT curing, both spontaneous and induced by bile, suggesting that the pSLT ccdAB genes may encode a homolog of the CcdAB addiction module previously described in the F sex factor. Unlike the F sex factor, synthesis of pSLT-encoded pili does not confer bile sensitivity. These observations may provide insights into the evolution of virulence plasmids in Salmonella subspecies I, as well as the causes of virulence plasmid loss in other Salmonella subspecies.     

Structural properties of periplasmic SodCI that correlate with virulence in Salmonella enterica serovar Typhimurium

Salmonella enterica strains survive and propagate in macrophages by both circumventing and resisting the antibacterial effectors normally delivered to the phagosome. An important aspect of Salmonella resistance is the production of periplasmic superoxide dismutase to combat phagocytic superoxide. S. enterica serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Both enzymes are produced during infection, but only SodCI contributes to virulence in the animal. Although 60% identical to SodCII at the amino acid level with very similar enzymatic properties, SodCI is dimeric, protease resistant, and tethered within the periplasm via a noncovalent interaction. In contrast, SodCII is monomeric and protease sensitive and is released from the periplasm normally by osmotic shock. We have constructed an enzymatically active monomeric SodCI enzyme by site-directed mutagenesis. The resulting protein was released by osmotic shock and sensitive to protease and could not complement the loss of wild-type dimeric SodCI during infection. To distinguish which property is most critical during infection, we cloned and characterized related SodC proteins from a variety of bacteria. Brucella abortus SodC was monomeric and released by osmotic shock but was protease resistant and could complement SodCI in the animal. These data suggest that protease resistance is a critical property that allows SodCI to function in the harsh environment of the phagosome to combat phagocytic superoxide. We propose a model to account for the various properties of SodCI and how they contribute to bacterial survival in the phagosome.     

Cyclic di-GMP signalling controls virulence properties of Salmonella enterica serovar Typhimurium at the mucosal lining

Cyclic di-GMP (c-di-GMP), a novel secondary signalling molecule present in most bacteria, controls transition between motility and sessility. In Salmonella enterica serovar Typhimurium ( S. typhimurium ) high c-di-GMP concentrations favour the expression of a biofilm state through expression of the master regulator CsgD. In this work, we investigate the effect of c-di-GMP signalling on virulence phenotypes of S. typhimurium. After saturation of the cell with c-di-GMP by overexpression of a di-guanylate cyclase, we studied invasion and induction of a pro-inflammatory cytokine in epithelial cells, basic phenotypes that are major determinants of S. typhimurium virulence. Elevated c-di-GMP had a profound effect on invasion into and IL-8 production by the gastrointestinal epithelial cell line HT-29. Invasion was mainly inhibited through CsgD and the extracellular matrix component cellulose, while inhibition of the pro-inflammatory response occurred through CsgD, which inhibited the secretion of monomeric flagellin. Our results suggest that transition between biofilm formation and virulence in S. typhimurium at the epithelial cell lining is mediated by c-di-GMP signalling through CsgD and cellulose expression.     

Porcine in vitro and in vivo models to assess the virulence of Salmonella enterica serovar Typhimurium for pigs

Salmonella Typhimurium infections in pigs pose an important human health hazard. One promising control measure is the development of live attenuated vaccine strains using defined knockout mutants. Preferably, screening of candidate knockout vaccine strains for attenuation should first be done in models allowing testing of a large number of strains. Thereafter, a limited number of selected strains should be further characterized in an experimental infection model in pigs. The aim of the present study was to develop such models. The invasive and proliferative characteristics of S. Typhimurium were assessed in both a non-polarized and a polarized porcine intestinal epithelial cell line. Neutrophils obtained from porcine blood were used to study the capacity of Salmonella to withstand killing by these phagocytes. The ability to induce an intestinal inflammatory response was investigated in a terminal intestinal loop model. The systemic phase of infection was mimicked by studying the uptake and intracellular survival of S. Typhimurium in porcine pulmonary alveolar macrophages and peripheral blood monocytes. These models should allow screening for attenuated strains. For further characterization, an experimental infection model was established, providing extensive data on the course of an oral infection and the optimal time points for colonization (day 5 postinoculation [pi]) and persistency (days 21-28 pi) in pigs. In conclusion, screening for virulence of S. Typhimurium strains with subsequent confirmation for a subset of strains in a well-defined experimental infection model would significantly reduce the number of experimental pigs required.     

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.     

Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium

Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.     

Bistability in myo-inositol utilization by Salmonella enterica serovar Typhimurium

The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.     

Impact of small RNA RaoN on nitrosative-oxidative stress resistance and virulence of Salmonella enterica serovar Typhimurium

RaoN is a Salmonella-specific small RNA that is encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11. We previously reported that RaoN is induced under conditions of acid and oxidative stress combined with nutrient limitation, contributing to the intramacrophage growth of Salmonella enterica serovar Typhimurium. However, the role of RaoN in nitrosative stress response and virulence has not yet been elucidated. Here we show that the raoN mutant strain has increased susceptibility to nitrosative stress by using a nitric oxide generating acidified nitrite. Extending previous research on the role of RaoN in oxidative stress resistance, we found that NADPH oxidase inhibition restores the growth of the raoN mutant in LPS-treated J774A.1 macrophages. Flow cytometry analysis further revealed that the inactivation of raoN leads to an increase in the intracellular level of reactive oxygen species (ROS) in Salmonella-infected macrophages, suggesting that RaoN is involved in the inhibition of NADPH oxidase-mediated ROS production by mechanisms not yet resolved. Moreover, we evaluated the effect of raoN mutation on the virulence in murine systemic infection and determined that the raoN mutant is less virulent than the wild-type strain following oral inoculation. In conclusion, small regulatory RNA RaoN controls nitrosative-oxidative stress resistance and is required for virulence of Salmonella in mice.     

Characterization of SrgA,a Salmonella enterica serovar Typhimurium virulence plasmid-encoded paralogue of the disulfide oxidoreductase DsbA,essential for biogenesis of plasmid-encoded fimbriae

Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of Salmonella enterica serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example, Escherichia coli, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family Enterobacteriaceae. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmid-encoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the S. enterica serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not. pefA and srgA were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in pef gene expression are also involved in srgA expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of S. enterica serovar Typhimurium.     

Immunoassay based on carbon nanotubes-enhanced ELISA for Salmonella enterica serovar Typhimurium

Among the methods used to detect pathogenic bacteria, enzyme linked immunosorbent assay (ELISA) is one of the most widely used techniques in routine sample analysis. For Salmonella enterica serovar Typhimurium detection, a typical ELISA yields a sensitivity of 10(6)-10(7)CFU/ml. To enhance the detection sensitivity, single-walled carbon nanotubes (SWCNTs) was employed in this study as a labelling platform for antibody and horseradish peroxidase (HRP) co-immobilizing. With high proteins recovery after the coupling process, the resulting Ab/SWCNTs/HRP bioconjugate was used in the proof-of-concept ELISA experiments. Limit of detection (LOD) was found to be 10(3) and 10(4)CFU/ml for direct and sandwich ELISA, respectively, when Ab/HRP at 1:400 ratio was used. This figure accounts for 1000-time greater in detection sensitivity when compared to a commercial Ab-HRP conjugate. The Ab/SWCNTs/HRP bioconjugate was tested further in real samples and found a superior activity over the commercial Ab-HRP by showing 100-time greater detection limit.