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Journal articlePollard DJ, Young JC, Covarelli V, et 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: 1098-5522Salmonella 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.
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Journal articleMavridou DAI, Gonzalez D, Clements A, et al., 2016,
The pUltra plasmid series: a robust and flexible tool for fluorescent labeling of Enterobacteria
, Plasmid, Vol: 87-88, Pages: 65-71, ISSN: 1095-9890Fluorescent 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.
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Journal articleGrundling A, Lee V, 2016,
Old concepts, new molecules and current approaches applied to the bacterial nucleotide signalling field
, Philosophical Transactions of the Royal Society B: Biological Sciences, Vol: 371, ISSN: 1471-2970Signalling nucleotides are key molecules that help bacteria to rapidly coordinate cellular pathways and adaptto changes in their environment. During the past ten years, the nucleotide-signalling field has seen muchexcitement, as several new signalling nucleotides have been discovered in both eukaryotic and bacterial cells.The fields have since advanced quickly, aided by the development of important tools such as the synthesis ofmodified nucleotides, which combined with sensitive mass spectrometry methods, allowed for the rapididentification of specific receptor proteins along with other novel genome-wide screening methods. In thisreview, we will describe the principle concepts of nucleotide signalling networks and summarize the recentwork that led to the discovery of the novel signalling nucleotides. We will also highlight current approachesapplied to the research in the field as well as resources and methodological advances aiding in a rapididentification of nucleotide specific receptor proteins.
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Journal articleCrepin VF, Collins JW, Habibzay M, et al., 2016,
Citrobacter rodentium mouse model of bacterial infection.
, Nature Protocols, Vol: 11, Pages: 1851-1876, ISSN: 1754-2189Infection of mice with Citrobacter rodentium is a robust model to study bacterial pathogenesis, mucosal immunology, the health benefits of probiotics and the role of the microbiota during infection. C. rodentium was first isolated by Barthold from an outbreak of mouse diarrhea in Yale University in 1972 and was 'rediscovered' by Falkow and Schauer in 1993. Since then the use of the model has proliferated, and it is now the gold standard for studying virulence of the closely related human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Here we provide a detailed protocol for various applications of the model, including bacterial growth, site-directed mutagenesis, mouse inoculation (from cultured cells and after cohabitation), monitoring of bacterial colonization, tissue extraction and analysis, immune responses, probiotic treatment and microbiota analysis. The main protocol, from mouse infection to clearance and analysis of tissues and host responses, takes ∼5 weeks to complete.
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Journal articleWilkinson RJ, Esmail H, Lesosky M, et al., 2016,
[18F]-FDG PET/CT characterisation of progressive HIV-associated tuberculosis
, Nature Medicine, ISSN: 1546-170XTuberculosis is classically divided into states of latent infection and active disease. Usingcombined positron emission and computed tomography in 35 asymptomatic, antiretroviraltherapy naïve, HIV-1 infected adults with latent tuberculosis, we identified ten individualswith pulmonary abnormalities suggestive of subclinical, active disease who weresignificantly more likely to progress to clinical disease. Our findings challenge theconventional two-state paradigm and may aid future identification of biomarkers predictiveof progression.
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Journal articleEsmail H, Lai RP, Lesosky M, et al., 2016,
Characterization of progressive HIV-associated tuberculosis using 2-deoxy-2-[(18)F]fluoro-D-glucose positron emission and computed tomography
, Nature Medicine, Vol: 22, Pages: 1090-1093, ISSN: 1546-170XTuberculosis is classically divided into states of latent infection and active disease. Using combined positron emission and computed tomography in 35 asymptomatic, antiretroviral-therapy-naive, HIV-1-infected adults with latent tuberculosis, we identified ten individuals with pulmonary abnormalities suggestive of subclinical, active disease who were substantially more likely to progress to clinical disease. Our findings challenge the conventional two-state paradigm and may aid future identification of biomarkers that are predictive of progression.
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Journal articleFurniss RCD, Slater S, Frankel G, et al., 2016,
Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signalling axis to prevent recycling endosome maturation during infection
, Journal of Molecular Biology, Vol: 428, Pages: 3399-3407, ISSN: 1089-8638Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate a plethora of host cell processes to establish infection of the gut mucosa. This manipulation is achieved via the injection of bacterial effector proteins into host cells using a Type III secretion system. We have previously reported that the conserved EHEC and EPEC effector EspG disrupts recycling endosome function, reducing cell surface levels of host receptors through accumulation of recycling cargo within the host cell. Here we report that EspG interacts specifically with the small GTPases ARF6 and Rab35 during infection. These interactions target EspG to endosomes and prevent Rab35-mediated recycling of cargo to the host cell surface. Furthermore, we show that EspG has no effect on Rab35-mediated uncoating of newly formed endosomes, and instead leads to the formation of enlarged EspG/TfR/Rab11 positive, EEA1/Clathrin negative stalled recycling structures. Thus, this paper provides a molecular framework to explain how EspG disrupts recycling whilst also reporting the first known simultaneous targeting of ARF6 and Rab35 by a bacterial pathogen.
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Journal articleGrundy GJ, Polo LM, Zeng Z, et al., 2016,
PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2).
, Nature Communications, Vol: 7, ISSN: 2041-1723PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.
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Journal articleSchuster C, Bellows L, Tosi T, et al., 2016,
The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus
, Science Signaling, Vol: 9, Pages: ra81-ra81, ISSN: 1945-0877Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S. aureus and found that the LAC*ΔgdpP strain of S. aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-β-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.
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Journal articleBaek K, Bowman L, Millership C, et al., 2016,
The cell wall polymer lipoteichoic acid becomes non-essential in Staphylococcus aureus cells lacking the ClpX chaperone
, mBio, Vol: 7, ISSN: 2150-7511Lipoteichoic acid is an essential component of the Staphylococcus aureus cell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus and vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth of S. aureus only as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes in S. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division in S. aureus may expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistant S. aureus.
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