Publications
Results
- Showing results for:
- Reset all filters
Search results
-
Journal articleGoddard AD, Bali S, Mavridou DAI, et 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: 1365-2958Nitrate 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 articleLee RBY, Mavridou DAI, Papadakos G, et al., 2016,
The uronic acid content of coccolith-associated polysaccharides provides insight into coccolithogenesis and past climate
, Nature Communications, Vol: 7, ISSN: 2041-1723Unicellular 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 articlePader V, Hakim S, Painter KL, et al., 2016,
Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids
, Nature Microbiology, Vol: 2, Pages: 1-8, ISSN: 2058-5276Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA)1,2. Although resistance is rare, treatment failure can occur in more than 20% of cases3,4 and so there is a pressing need to identify and mitigate factors that contribute to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. Wild-type S. aureus was killed rapidly by daptomycin, but Agr-defective mutants survived antibiotic exposure by releasing membrane phospholipids, which bound and inactivated the antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that protects against daptomycin, which can be compromised by Agr-triggered toxin production or an existing therapeutic antibiotic.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
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.