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.     

Either periplasmic tethering or protease resistance is sufficient to allow a SodC to protect Salmonella enterica serovar Typhimurium from phagocytic superoxide

Salmonella Typhimurium combats phagocytic superoxide by producing the periplasmic superoxide dismutase, SodCI. The homologous protein, SodCII, is also produced during infection, but does not contribute to virulence. The proteins physically differ in that SodCI is dimeric, protease resistant and non-covalently tethered within the periplasm. Conversely, SodCII is a protease-sensitive monomer that is released normally from the periplasm by osmotic shock. To identify which properties correlate with virulence, we constructed over 20 enzymatically functional hybrid SodC proteins and assayed them for protease susceptibility, release by osmotic shock, multimerization and affinity for metal cofactors. Protease susceptibility maps to the C-terminus of SodCII, while SodCI residues 120-131 are required for tethering. A protease-resistant SodCII hybrid was able to substitute for SodCI during infection. Interestingly, a tethered but protease-sensitive SodCII hybrid was also able to confer protection. Thus, either tethering or protease resistance is sufficient for a SodC to function during infection. These results support our model that in the macrophage, the outer membrane of Salmonella is partially disrupted by antimicrobial peptides. Periplasmic proteins, including SodCII, are released and/or phagocytic proteases gain access. SodCI is both tethered within the periplasm and protease resistant, thereby surviving to detoxify superoxide.     

SigE is a chaperone for the Salmonella enterica serovar Typhimurium invasion protein SigD

SigD is translocated into eucaryotic cells by a type III secretion system. In this work, evidence that the putative chaperone SigE directly interacts with SigD is presented. A bacterial two-hybrid system demonstrated that SigE can interact with itself and SigD. In addition, SigD was specifically copurified with SigE-His(6) on a nickel column.     

ZntB is a novel Zn2+ transporter in Salmonella enterica serovar Typhimurium

A Zn2+ transport system encoded by the zntB locus of Salmonella enterica serovar Typhimurium has been identified. The protein encoded by this locus is homologous to the CorA family of Mg2+ transport proteins and is widely distributed among the eubacteria. Mutations at zntB confer an increased sensitivity to the cytotoxic effects of Zn2+ and Cd2+, a phenotype that suggests that the encoded protein mediates the efflux of both cations. A direct analysis of transport activity identified a capacity for Zn2+ efflux. These data identify ZntB as a zinc efflux pathway in the enteric bacteria and assign a new function to the CorA family of cation transporters.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

Salmonella enterica serovar Typhimurium DT104 displays a rugose phenotype

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25 degrees C after 3 days of incubation. Incubation at 37 degrees C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25 degrees C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.     

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.     

Differences in enzymatic properties allow SodCI but not SodCII to contribute to virulence in Salmonella enterica serovar Typhimurium strain 14028

Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth. By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter. In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is "tethered" within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.     

The apeE gene of Salmonella enterica serovar Typhimurium is induced by phosphate limitation and regulated by phoBR

Mutations in apeR, a regulatory locus of the outer membrane esterase apeE from Salmonella enterica serovar Typhimurium, were shown to be alleles of the pstSCAB-phoU high-affinity phosphate transport operon. Expression of apeE was induced by phosphate limitation, and this induction required the phoBR phosphate regulatory system.     

Hydrogen-stimulated carbon acquisition and conservation in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium can utilize molecular hydrogen for growth and amino acid transport during anaerobic growth. Via microarray we identified H(2) gas-affected gene expression changes in Salmonella. The addition of H(2) caused altered expression of 597 genes, of which 176 genes were upregulated and 421 were downregulated. The significantly H(2)-upregulated genes include those that encode proteins involved in the transport of iron, manganese, amino acids, nucleosides, and sugars. Genes encoding isocitrate lyase (aceA) and malate synthase (aceB), both involved in the carbon conserving glyoxylate pathway, and genes encoding the enzymes of the d-glucarate and d-glycerate pathways (gudT, gudD, garR, garL, garK) are significantly upregulated by H(2). Cells grown with H(2) showed markedly increased AceA enzyme activity compared to cells without H(2). Mutant strains with deletion of either aceA or aceB had reduced H(2)-dependent growth rates. Genes encoding the glutamine-specific transporters (glnH, glnP, glnQ) were upregulated by H(2), and cells grown with H(2) showed increased [(14)C]glutamine uptake. Similarly, the mannose uptake system genes (manX, manY) were upregulated by H(2,) and cells grown with H(2) showed about 2.0-fold-increased [(14)C]d-mannose uptake compared to the cells grown without H(2). Hydrogen stimulates the expression of genes involved in nutrient and carbon acquisition and carbon-conserving pathways, linking carbon and energy metabolism to sustain H(2)-dependent growth.     

Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium SL1344 in mice virulence

opgB gene of Salmonella enterica serovar Typhimurium was identified earlier in a genome-wide screen for mice virulence (Valentine et al. in Infect Immun 66:3378-3383, 1998). Although mutation in opgB resulted in avirulent Salmonella strain, how this gene contributes to pathogenesis remains unclear. Based on DNA homology, opgB is predicted to be responsible for adding phosphoglycerate residues to osmoregulated periplasmic glucans (OPGs) giving them anionic characteristics. In Escherichia coli, yet another gene, opgC, is also reported to contribute to anionic characteristics of OPGs by adding succinic acid residues. We constructed opgB, opgC, and opgBC double mutants of S. enterica serovar Typhimurium strain SL1344. As predicted opgBC mutant synthesized neutral OPGs that were devoid of any anionic substituents. However, opgB, opgC, and opgBC mutations had no significant impact on mice virulence as well as on competitive organ colonization. In low osmotic conditions, opgB, opgC, and opgBC mutants exhibited delay in growth initiation in the presence of sodium deoxycholate. Anionic substituents of OPGs from Salmonella although appear to be needed to overcome resistance of deoxycholate in hypoosmotic growth media, no evidence was found for their role in mice 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.     

Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 10(9) CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 10(4) CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 10(6) CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.