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  • Journal article
    Bowman L, Zeden MS, Schuster CF, Kaever V, Grundling Aet al., 2016,

    New Insights into the Cyclic di-Adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic-Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

    , Journal of Biological Chemistry, Vol: 291, Pages: 26970-26986, ISSN: 1083-351X

    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane bound Asp-His-His and Asp-His-His associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared to the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus, but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.

  • Journal article
    Bayer Santos E, Durkin CH, Rigano L, Kupz A, Alix E, Cerny O, Jennings E, Liu M, Ryan A, Lapaque N, Kaufmann S, Holden D, Robb Cet al., 2016,

    The Salmonella effector SteD mediates MARCH8-1 dependent ubiquitination of MHC II molecules and inhibits T cell activation

    , Cell Host & Microbe, Vol: 20, Pages: 584-595, ISSN: 1934-6069

    The SPI-2 type III secretion system (T3SS) of intracellular Salmonella enterica translocates effector proteins into mammalian cells. Infection of antigen-presenting cells results in SPI-2 T3SS-dependent ubiquitination and reduction of surface-localized mature MHC class II (mMHCII). We identify the effector SteD as required and sufficient for this process. In Mel Juso cells, SteD localized to the Golgi network and vesicles containing the E3 ubiquitin ligase MARCH8 and mMHCII. SteD caused MARCH8-dependent ubiquitination and depletion of surface mMHCII. One of two transmembrane domains and the C-terminal cytoplasmic region of SteD mediated binding to MARCH8 and mMHCII, respectively. Infection of dendritic cells resulted in SteD-dependent depletion of surface MHCII, the co-stimulatory molecule B7.2, and suppression of T cell activation. SteD also accounted for suppression of T cell activation during Salmonella infection of mice. We propose that SteD is an adaptor, forcing inappropriate ubiquitination of mMHCII by MARCH8 and thereby suppressing T cell activation.

  • Journal article
    Ale A, Crepin VF, Collins, Constantinou N, Habibzay, Babtie AC, Frankel G, Stumpf MPet al., 2016,

    Model of host-pathogen Interaction dynamics links In vivo optical imaging and immune responses

    , Infection and Immunity, Vol: 85, ISSN: 1098-5522

    Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.

  • Journal article
    Thurston T, Matthews S, Jennings E, Alix E, Shao F, Shenoy A, Birrell M, Holden Det al., 2016,

    Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death

    , Nature Communications, Vol: 7, ISSN: 2041-1723

    Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria.

  • Journal article
    Grabe GJ, Zhang Y, Przydacz M, Rolhion N, Yang Y, Pruneda JN, Komander D, Holden DW, Hare SAet al., 2016,

    The Salmonella effector SpvD is a cysteine hydrolase with a serovar-specific polymorphism influencing catalytic activity, suppression of immune responses and bacterial virulence

    , Journal of Biological Chemistry, Vol: 291, Pages: 25853-25863, ISSN: 1083-351X

    Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising C73, H162, and D182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro. A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host.

  • Journal article
    Goddard AD, Bali S, Mavridou DAI, Luque- Almagro VM, Gates AJ, Roldán MD, Newstead S, Richardson DJ, Ferguson SJet al., 2016,

    The Paracoccus denitrificans NarK-like nitrate and nitrite transporters; probing nitrate uptake and nitrate/nitrite exchange mechanisms

    , Molecular Microbiology, Vol: 103, Pages: 117-133, ISSN: 1365-2958

    Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange.

  • Journal article
    Lee RBY, Mavridou DAI, Papadakos G, McClelland HLO, Rickaby REMet al., 2016,

    The uronic acid content of coccolith-associated polysaccharides provides insight into coccolithogenesis and past climate

    , Nature Communications, Vol: 7, ISSN: 2041-1723

    Unicellular phytoplanktonic algae (coccolithophores) are among the most prolific producers of calcium carbonate on the planet, with a production of ∼1026 coccoliths per year. During their lith formation, coccolithophores mainly employ coccolith-associated polysaccharides (CAPs) for the regulation of crystal nucleation and growth. These macromolecules interact with the intracellular calcifying compartment (coccolith vesicle) through the charged carboxyl groups of their uronic acid residues. Here we report the isolation of CAPs from modern day coccolithophores and their prehistoric predecessors and we demonstrate that their uronic acid content (UAC) offers a species-specific signature. We also show that there is a correlation between the UAC of CAPs and the internal saturation state of the coccolith vesicle that, for most geologically abundant species, is inextricably linked to carbon availability. These findings suggest that the UAC of CAPs reports on the adaptation of coccolithogenesis to environmental changes and can be used for the estimation of past CO2 concentrations.

  • Journal article
    Pader V, Hakim S, Painter KL, Wigneshweraraj S, Clarke TB, Edwards Aet al., 2016,

    Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids

    , Nature Microbiology, Vol: 2, ISSN: 2058-5276
  • Journal article
    Pollard DJ, Young JC, Covarelli V, Herrera-León S, Connor TR, Fookes M, Walker D, Echeita A, Thomson NR, Berger CN, Frankel Get al., 2016,

    The type III secretion system effector SeoC of Salmonella enterica subspecies salamae and arizonae ADP-ribosylates Src and inhibits opsono-phagocytosis

    , Infection and Immunity, Vol: 84, Pages: 3618-3628, ISSN: 1098-5522

    Salmonella spp. utilize type III secretion systems (T3SS) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study we compared a draft genome assembly of S. enterica subsp. salamae strain 3588/07 (S. salamae) against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and S. bongori strain 12419. S. salamae encode the Salmonella pathogenicity island (SPI)-1; SPI-2 and the locus of enterocyte effacement (LEE) T3SSs. Though several key S. Typhimurium effector genes are missing (e.g. avrA, sopB and sseL), S. salamae invades HeLa cells and contain homologues of S. bongori sboK and sboC, which we named seoC. SboC and SeoC are homologues of EspJ from enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), which inhibits Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates we identified EspJ homologues in S. bongori, S. salamae and S. enterica subsp. arizonae (S. arizonae). The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro. Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized bead into Cos-7 cells stably expressing GFP-FcγRIIa. Concurrently, S. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC dependent manner. These results show that S. bongori, S. salamae and S. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and sheds light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.

  • Journal article
    Mavridou DAI, Gonzalez D, Clements A, Foster KRet al., 2016,

    The pUltra plasmid series: a robust and flexible tool for fluorescent labeling of Enterobacteria

    , Plasmid, Vol: 87-88, Pages: 65-71, ISSN: 1095-9890

    Fluorescent labeling has been an invaluable tool for the study of living organisms andbacterial species are no exception to this. Here we present and characterize the pUltraplasmids which express constitutively a fluorescent protein gene (GFP, RFP, YFP or CFP)from a strong synthetic promoter and are suitable for the fluorescent labeling of a broad rangeof Enterobacteria. The amount of expressed fluorophore from these genetic constructs issuch, that the contours of the cells can be delineated on the basis of the fluorescent signalonly. In addition, labeling through the pUltra plasmids can be used successfully forfluorescence and confocal microscopy while unambiguous distinction of cells labeled withdifferent colors can be carried out efficiently by microscopy or flow cytometry. We comparethe labeling provided by the pUltra plasmids with that of another plasmid series encodingfluorescent proteins and we show that the pUltra constructs are vastly superior in signalintensity and discrimination power without having any detectable growth rate effects for thebacterial population. We also use the pUltra plasmids to produce mixtures of differentiallylabeled pathogenic Escherichia, Shigella and Salmonella species which we test duringinfection of mammalian cells. We find that even inside the host cell, different strains can bedistinguished effortlessly based on their fluorescence. We, therefore, conclude that the pUltraplasmids are a powerful labeling tool especially useful for complex biological experimentssuch as the visualization of ecosystems of different bacterial species or of enteric pathogensin contact with their hosts.

This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.

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