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.     

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.     

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.     

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.     

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.     

Structural and functional characterization of SiiA,an auxiliary protein from the SPI4‐encoded type 1 secretion system from Salmonella enterica

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)‐encoded type one secretion system (T1SS) and the SPI1‐encoded type three secretion system (T3SS‐1). The SPI4‐encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure–function relationships in SiiA, a non‐canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA‐PD). The crystal structure of SiiA‐PD displays homology to that of MotB and other peptidoglycan (PG)‐binding domains. SiiA‐PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA‐MotB homology is paralleled by a predicted SiiB‐MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.     

The Salmonella effector SteA binds phosphatidylinositol 4‐phosphate for subcellular targeting within host cells

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4‐phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella‐containing vacuole (SCV) and to Salmonella‐induced tubules; using the PI(4)P‐binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N‐terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.     

Immunity to Salmonella from a dendritic point of view

Dendritic cells (DC) are the key link between innate and adaptive immunity. Features of DC, including their presence at sites of antigen entry, their ability to migrate from peripheral sites to secondary lymphoid organs, and their superior capacity to stimulate naïve T cells places them in this pivotal role in the immune system. DC also produce cytokines, particularly IL‐12, upon antigen encounter and can thus influence the ensuing adaptive immune response. As DC are phagocytic antigen‐presenting cells located at sites exposed to bacterial invaders, studies have been performed to gain insight into the role of DC in combating bacterial infections. Indeed, studies with Salmonella have shown that DC can internalize and process this bacterium for peptide presentation on MHC‐II as well as MHC‐I. DC can also act as bystander antigen‐­presenting cells by presenting Salmonella antigens after internalizing neighbouring cells that have undergone Salmonella‐induced apoptotic death. DC also produce IL‐12 and TNF‐α upon Salmonella encounter. Moreover, studies in a murine infection model have shown that splenic DC increase surface expression of co‐stimulatory molecules during infection, and DC contain intracellular bacteria. In addition, quantitative changes occur in splenic DC numbers in the early stages of oral Salmonella infection, and this is accompanied by redistribution of the defined DC subsets in the spleen of infected mice. DC from Salmonella‐infected mice also produce cytokines and can stimulate bacteria‐specific T cells upon ex vivo co‐culture. In addition, DC may play a role in the traversal of bacteria from the intestinal lumen. Studying the function of DC during Salmonella infection provides insight into the capacity of this sophisticated antigen‐presenting cell to initiate and modulate the immune response to bacteria.     

MUC1 mediates Pneumocystis murina binding to airway epithelial cells

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defence during airway infection. MUC1 is the best characterised membrane‐tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Overexpression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knockdown of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma‐derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labelling indicated that MUC1 and Pneumocystis were co‐localised in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knockdown of MUC1 expression by siRNA. Pneumocystis caused increased IL‐6 and IL‐8 secretion by A549 cells, and knockdown of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.     

The Intimin periplasmic domain mediates dimerisation and binding to peptidoglycan

Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C‐terminal extracellular domain and a β‐barrel transmembrane domain, both proteins also contain a short N‐terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α‐helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM‐containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

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.     

The Salmonella Typhimurium effector protein SopE transiently localizes to the early SCV and contributes to intracellular replication

Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non‐phagocytic cells by a Type III secretion system (TTSS) and cognate effector proteins. Upon host cell entry, S. Tm expresses a second TTSS and subverts intracellular trafficking to create a replicative niche – the Salmonella‐containing vacuole (SCV). SopE, a guanidyl exchange factor (GEF) for Rac1 and Cdc42, is translocated by the TTSS‐1 upon host cell contact and promotes entry through triggering of actin‐dependent ruffles. After host cell entry, the bulk of SopE undergoes proteasomal degradation. Here we show that a subfraction is however detectable on the nascent SCV membrane up to ～ 6 h post infection. Membrane localization of SopE and the closely related SopE2 differentially depend on the Rho‐GTPase‐binding GEF domain, and to some extent involves also the unstructured N‐terminus. SopE localizes transiently to the early SCV, dependent on continuous synthesis and secretion by the TTSS‐1 during the intracellular state. Mutant strains lacking SopE or SopE2 are attenuated in early intracellular replication, while complementation restores this defect. Hence, the present study reveals an unanticipated role for SopE and SopE2 in establishing the Salmonella replicative niche, and further emphasizes the importance of entry effectors in later stages of host‐cell manipulation.     

Synthesis and biological evaluation of new active For‐Met‐Leu‐Phe‐OMe analogues containing para‐substituted Phe residues

In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH₃ and ‐C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The Annexin A2/p11 complex is required for efficient invasion of Salmonella Typhimurium in epithelial cells

The facultative intracellular pathogen, Salmonella enterica, triggers its own uptake into non‐phagocytic epithelial cells. Invasion is dependent on a type 3 secretion system (T3SS), which delivers a cohort of effector proteins across the plasma membrane where they induce dynamic actin‐driven ruffling of the membrane and ultimately, internalization of the bacteria into a modified phagosome. In eukaryotic cells, the calcium‐ and phospholipid‐binding protein Annexin A2 (AnxA2) functions as a platform for actin remodelling in the vicinity of dynamic cellular membranes. AnxA2 is mostly found in a stable heterotetramer, with p11, which can interact with other proteins such as the giant phosphoprotein AHNAK. We show here that AnxA2, p11 and AHNAK are required for T3SS‐mediated Salmonella invasion of cultured epithelial cells and that the T3SS effector SopB is required for recruitment of AnxA2 and AHNAK to Salmonella invasion sites. Altogether this work shows that, in addition to targeting Rho‐family GTPases, Salmonella can intersect the host cell actin pathway via AnxA2.