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.     

Periplasmic superoxide dismutase SodCI of Salmonella binds peptidoglycan to remain tethered within the periplasm

Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell‐associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non‐covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI and allow the now tethered SodCII to function during infection.     

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.     

Differences in gene expression levels and in enzymatic qualities account for the uneven contribution of superoxide dismutases SodCI and SodCII to pathogenicity in Salmonella enterica

Most Salmonella enterica serovars produce two periplasmic [Cu,Zn] superoxide dismutases, SodCI, which is prophage encoded, and SodCII, encoded by a conserved chromosomal gene. Both enzymes were proposed to enhance Salmonella virulence by protecting bacteria against products of macrophage oxidative burst. However, we previously found SodCI, but not SodCII, to play a role during mouse infection by S. enterica serovar Typhimurium. Here we have extended these findings to another serovar of epidemiological relevance: sv Enteritidis. In both serovars, the dominant role of SodCI in virulence correlates with its higher levels in bacteria proliferating in mouse tissues, relative to SodCII. To analyze the basis of these differences, the coding sequences of sodCI and sodCII genes were exchanged with the reciprocal 5'-regions (in serovar Typhimurium). The accumulation patterns of the two proteins in vivo were reversed as a result, indicating that the regulatory determinants lie entirely within the regions upstream from the initiation codon. In the construct with the sodCI gene fused to the sodCII 5'-region, SodCI contribution to virulence was reduced but remained significant. Thus, both, high-level expression and some unidentified qualities of the enzyme participate in the phenotypic dominance of SodCI over SodCII in Salmonella pathogenicity.     

Differential accumulation of Salmonella[Cu,Zn] superoxide dismutases SodCI and SodCII in intracellular bacteria: correlation with their relative contribution to pathogenicity

Most Salmonella enterica strains have two peri-plasmic [Cu, Zn] superoxide dismutases, SodCI and SodCII, encoded by prophage and chromosomal genes respectively. Both enzymes are thought to play a role in Salmonella pathogenicity by intercepting reactive oxygen species produced by the host's innate immune response. To examine the apparent redundancy, we have compared the levels of epitope-tagged SodCI and SodCII proteins in bacteria growing in vitro, as well as inside tissue culture cells and in mouse tissues. Concomitantly, we have measured the abilities of mutants of either or both sodC genes to proliferate in infected mice in competition assays. Our results show a striking variation in the relative abundance of the two proteins in different environments. In vitro, both proteins accumulate when bacteria enter stationary phase; however, the increase is much sharper and conspicuous for SodCII than for SodCI. In contrast, SodCI vastly predominates in intracellular bacteria where SodCII levels are negligible. In agreement with these findings, most, if not all, of the contribution of [Cu, Zn] superoxide dismutase activity to murine salmonellosis can be ascribed to the SodCI protein. Overall the results of this work suggest that the duplicate sodC genes of Salmonella have evolved to respond to different sets of conditions encountered by bacteria inside the host and in the environment.     

Identification of GtgE,a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium

The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.     

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis

Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC , encoding [Cu,Zn]-SOD, from Salmonella typhimurium . The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC , in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S . typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S . typhimurium , S . choleraesuis and S . dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro , perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella , the most efficient overwhelming the proposed protective effect of periplasmic SOD.     

渗压震扰法释放Aeromonas sp.2016菌株外周间质中几丁质酶的研究

气单胞菌Aeromonassp．2016菌株能产生多种几丁质酶,其中的胞外酶C可能聚集于细胞外周胞质。为了避免破碎菌体而产生过多的杂蛋白,探索了用渗压震扰法(osmoticshock)来释放这部分酶。主要步骤是:先将菌体悬浮在20％蔗糖-0．03mol／LTris-HCI(pH8．0)高渗透压的溶液中,再快速转移到纯水低渗透压溶液中,产生瞬间渗压震荡,释放细胞外周胞质中的酶。结果表明,通过渗压震扰法释放出的酶纯度最高,比活力达到142．79U／g,比培养液上清液的54．46U／g和菌体破碎样品的14．66U／g分别高1．6倍和8．7倍,可用于纯化目的蛋白。     

Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli.

Bacterial expression systems can greatly facilitate protein engineering of antibodies. We have developed a system for high-level expression of antibodies, antibody fragments, or hybrid antibodies with novel effector functions in the periplasm of Escherichia coli. From 5 ml of cells, a simple extraction yields sufficient material for SDS-gel electrophoresis, detection and characterization of hapten binding. To demonstrate our system, heavy-chain variable regions and lambda 1 light chains of a mouse anti-NP antibody were synthesized as hybrid proteins with a bacterial signal peptide (Omp F). Each chain is secreted into the periplasm where processing (cleavage of the signal peptide), folding and heterodimer association take place. Periplasmic proteins are released by cold osmotic shock, and hapten-binding activity is easily detected without further manipulation. The ease of genetic engineering in this system will facilitate the production of immunoglobulin derivatives designed for specific applications, and expression of these molecules in a native state will allow the rapid screening of combinatorial libraries and the results of mutagenesis.     

Responses to reactive oxygen intermediates and virulence of Salmonella typhimurium

Salmonella typhimurium is an intracellular pathogen that can survive and replicate in macrophages. One of the host defense mechanisms that S. typhimurium encounters upon infection is superoxide produced by the phagocytes' NADPH-oxidase. Salmonella has evolved numerous ways of coping with superoxide in the extracellular environment. In addition, Salmonella has to defend itself against superoxide produced as a by-product of aerobic respiration. Over the last decade, research on bacterial mutants has led to the identification of Salmonella strains that differ from their parental strain in susceptibility to superoxide in vitro. However, the consequences of such mutations for bacterial virulence are highly variable, indicating that superoxide sensitivity per se is not a characteristic that renders Salmonella less virulent. By discussing various bacterial mutants classified according to their in vitro sensitivity to superoxide, we will exemplify the complex mechanisms that Salmonella has evolved to cope with superoxide stress.     

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.     

N-Terminal clustering of the O-glycosylation sites in the Mycobacterium tuberculosis lipoprotein SodC

SodC is one of two superoxide dismutases produced by Mycobacteriumtuberculosis. This protein was previously shown to contributeto virulence and to act as a B-cell antigen. SodC is also aputative lipoprotein, and like other Sec-translocated mycobacterialproteins it was suggested to be modified with glycosyl units.To definitively define the glycosylation of SodC, we appliedan approach that combined site-directed mutagenesis, lectinbinding, and mass spectrometry. This resulted in identificationof six O-glycosylated residues within a 13-amino-acid regionnear the N-terminus. Each residue was modified with one to threehexose units, and the most dominant SodC glycoform was modifiedwith nine hexose units. In addition to O-glycosylation of threonineresidues, this study provides the first evidence of serine O-glycosylationin mycobacteria. When combined with bioinformatic analyses,the clustering of O-glycosylation appeared to occur in a regionof SodC with a disordered structure and not in regions importantto the enzymatic activity of SodC. The use of recombinant aminoacid substitutions to alter glycosylation sites provided furtherevidence that glycosylation influences proteolytic processingand ultimately positioning of cell wall proteins.     

Inducible prophages contribute to Salmonella virulence in mice

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.     

Copper,Zinc-Superoxide Dismutase from Clinically Isolated <Emphasis Type="Italic">Escherichia coli</Emphasis>: Cloning,Analysis of <Emphasis Type="Italic">sodC</Emphasis> and Its Possible Role in Pathogenicity

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.     

A fresh view of the cell biology of copper in enterobacteria

Copper ions are essential but also very toxic. Copper resistance in bacteria is based on export of the toxic ion, oxidation from Cu(I) to Cu(II), and sequestration by copper‐binding metal chaperones, which deliver copper ions to efflux systems or metal‐binding sites of copper‐requiring proteins. In their publication in this issue, Osman et al. ( 2013 ) demonstrate how tightly copper resistance, homeostasis and delivery pathways are interwoven in Salmonella enterica sv. Typhimurium. Copper is transported from the cytoplasm by the two P‐type ATPases CopA and GolT to the periplasm and transferred to SodCII by CueP, a periplasmic copper chaperone. When copper levels are higher, SodCII is also able to bind copper without the help of CueP. This scheme raises the question as to why copper ions present in the growth medium have to make the detour through the cytoplasm. The data presented in the publication by Osman et al. ( 2013 ) change our view of the cell biology of copper in enterobacteria.     

Direct ROS Scavenging Activity of CueP from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that has evolved to survive in the phagosome of macrophages. The periplasmic copper-binding protein CueP was initially known to confer copper resistance to S. Typhimurium. Crystal structure and biochemical studies on CueP revealed a putative copper binding site surrounded by the conserved cysteine and histidine residues. A recent study reported that CueP supplies copper ions to periplasmic Cu, Zn-superoxide dismutase (SodCII) at a low copper concentration and thus enables the sustained SodCII activity in the periplasm. In this study, we investigated the role of CueP in copper resistance at a high copper concentration. We observed that the survival of a cueP-deleted strain of Salmonella in macrophage phagosome was significantly reduced. Subsequent biochemical experiments revealed that CueP specifically mediates the reduction of copper ion using electrons released during the formation of the disulfide bond. We observed that the copper ion-mediated Fenton reaction in the presence of hydrogen peroxide was blocked by CueP. This study provides insight into how CueP confers copper resistance to S. Typhimurium in copper-rich environments such as the phagosome of macrophages.