121 results on '"Mavridou, Despoina"'
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2. Automated sample preparation with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and iodoacetamide derivatization reagents for enantioselective liquid chromatography tandem mass spectrometry amino acid analysis
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Li, Feiyang, Karongo, Ryan, Mavridou, Despoina, Horak, Jeannie, Sievers-Engler, Adrian, and Lämmerhofer, Michael
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- 2023
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3. A novel stabilization mechanism for the type VI secretion system sheath
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Bernal, Patricia, Furniss, R. Christopher D., Fecht, Selina, Leung, Rhoda C. Y., Spiga, Livia, Mavridou, Despoina A. I., and Filloux, Alain
- Published
- 2021
4. Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells
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Nolan, Laura M., Cain, Amy K., Clamens, Thomas, Furniss, R. Christopher D., Manoli, Eleni, Sainz-Polo, Maria A., Dougan, Gordon, Albesa-Jové, David, Parkhill, Julian, Mavridou, Despoina A. I., and Filloux, Alain
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- 2021
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5. Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.
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Zhai, Ziran, Mavridou, Despoina, Damian, Matteo, Mutti, Francesco G., Schoenmakers, Peter J., and Gargano, Andrea F. G.
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- 2024
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6. The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors
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Pissaridou, Panayiota, Allsopp, Luke P., Wettstadt, Sarah, Howard, Sophie A., Mavridou, Despoina A. I., and Filloux, Alain
- Published
- 2018
7. Droplet printing reveals the importance of micron-scale structure for bacterial ecology
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Krishna Kumar, Ravinash, Meiller-Legrand, Thomas A., Alcinesio, Alessandro, Gonzalez, Diego, Mavridou, Despoina A. I., Meacock, Oliver J., Smith, William P. J., Zhou, Linna, Kim, Wook, Pulcu, Gökçe Su, Bayley, Hagan, and Foster, Kevin R.
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- 2021
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8. Making the Best of Aggression: The Many Dimensions of Bacterial Toxin Regulation
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Gonzalez, Diego and Mavridou, Despoina A.I.
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- 2019
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9. Costs and benefits of provocation in bacterial warfare
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Gonzalez, Diego, Sabnis, Akshay, Foster, Kevin R., and Mavridou, Despoina A. I.
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- 2018
10. Elucidation of the structure-function relationships in the bacterial transmembrane disulfide oxidoreductase DsbD
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Mavridou, Despoina A. I.
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572.6 - Published
- 2008
11. Chapter Five - Making a chink in their armor: Current and next-generation antimicrobial strategies against the bacterial cell envelope.
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Kadeřábková, Nikol, Mahmood, Ayesha J. S., Furniss, R. Christopher D., and Mavridou, Despoina A. I.
- Abstract
Gram-negative bacteria are uniquely equipped to defeat antibiotics. Their outermost layer, the cell envelope, is a natural permeability barrier that contains an array of resistance proteins capable of neutralizing most existing antimicrobials. As a result, its presence creates a major obstacle for the treatment of resistant infections and for the development of new antibiotics. Despite this seemingly impenetrable armor, in-depth understanding of the cell envelope, including structural, functional and systems biology insights, has promoted efforts to target it that can ultimately lead to the generation of new antibacterial therapies. In this article, we broadly overview the biology of the cell envelope and highlight attempts and successes in generating inhibitors that impair its function or biogenesis. We argue that the very structure that has hampered antibiotic discovery for decades has untapped potential for the design of novel next-generation therapeutics against bacterial pathogens. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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12. Making a chink in their armor: Current and next-generation antimicrobial strategies against the bacterial cell envelope
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Kadeřábková, Nikol, Mahmood, Ayesha J.S., Furniss, R. Christopher D., and Mavridou, Despoina A.I.
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- 2023
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13. Carbapenem-Resistant and ESBL-Producing Enterobacterales Emerging in Central Texas.
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Parker, Jennifer K, Gu, Richard, Estrera, Gregory A, Kirkpatrick, Betsy, Rose, Dusten T, Mavridou, Despoina AI, Mondy, Kristin E, and Davies, Bryan W
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WHOLE genome sequencing ,MICROBIAL sensitivity tests ,INFECTION prevention ,DRUG resistance in bacteria ,KLEBSIELLA pneumoniae - Abstract
Purpose: Carbapenem-resistant Enterobacterales (CRE) are subject to intense global monitoring in an attempt to maintain awareness of prevalent and emerging resistance mechanisms and to inform treatment and infection prevention strategies. CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are not usually examined collectively in regards to their shared pool of resistance determinants. Here, we genetically and phenotypically assess clinical isolates of CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in the growing region of Central Texas, where CRE are emergent and occurrence of non-carbapenemase-producing-CRE (non-CP-CRE) infections is increasing. Methods: CRE (n=16) and ESBL-producing Enterobacterales (n=116) isolates were acquired from a regional hospital in Central Texas between December 2018 and January 2020. Isolates were assessed genetically and phenotypically using antibiotic susceptibility testing, targeted PCR, and whole genome sequencing. Results: CRE infections are increasing in incidence in Central Texas, and Klebsiella pneumoniae is causing the majority of these infections. Moreover, K. pneumoniae sequence type (ST) 307 is commonly found among both non-CP-CRE and EBSL-producing strains. Isolates carry similar plasmids harboring the gene for the ESBL CTX-M-15 and belong to the global lineage, rather than the Texas lineage, of ST307. Antibiotic resistance profiles, sequence data, and clinical records suggest that porin mutations may promote the transition of ST307 isolates from ESBL-producing to non-CP-CRE. In addition to antibiotic resistance mechanisms, several CRE isolates harbor active colicinogenic plasmids, which might influence the competitiveness of these bacteria during patient colonization. Conclusion: K. pneumoniae of the global ST307 lineage is circulating in Central Texas and is responsible for both non-CP CRE and ESBL-producing Enterobacterales infections. Enhanced surveillance is needed to understand the possible routes for the emergence of non-CP-CRE from EBSL-producing strains. [ABSTRACT FROM AUTHOR]
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- 2023
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14. Control of Periplasmic Interdomain Thiol:Disulfide Exchange in the Transmembrane Oxidoreductase DsbD
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Mavridou, Despoina A.I., Stevens, Julie M., Goddard, Alan D., Willis, Antony C., Ferguson, Stuart J., and Redfield, Christina
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- 2009
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15. 1H, 13C and 15N resonance assignments for the oxidized and reduced states of the N-terminal domain of DsbD from Escherichia coli
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Mavridou, Despoina A. I., Stelzl, Lukas S., Ferguson, Stuart J., and Redfield, Christina
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- 2012
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16. Staphylococcal DNA repair confers tolerance of the bactericidal activity of both neutrophils and antibiotics
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Ha, Kam Pou, Clarke, Rebecca S., Brittan, Jane L., Rowley, Jessica E., Mavridou, Despoina A. I., Clarke, Thomas B., Nobbs, Angela H., and Edwards, Andrew M.
- Abstract
Staphylococcus aureus is a leading cause of chronic and recurrent infections of the skin, bones, joints and bloodstream. During infection, S. aureus faces the twin threats of the host immune system and therapeutic antibiotics. Since both host immune cells and antibiotics generate reactive oxygen species, we hypothesised that S. aureus may employ a common mechanism to repair damage caused by either threat. We found that staphylococcal DNA is damaged during exposure to both human neutrophils and most bactericidal antibiotics. To understand the nature of this damage and how S. aureus repairs it, we screened a panel of transposon mutants defective for various DNA repair processes. This revealed that loss of the rexBA operon significantly reduced staphylococcal survival in human blood, during incubation with purified neutrophils, in the peritoneal cavity of mice and during exposure to a large panel of bactericidal antibiotics. We then used biochemical assays to demonstrate that RexAB is a member of the AddAB family of ATP-dependent helicase/nucleases that are required for the repair of DNA double strand breaks. Finally, we found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA repair constitutes a broadly conserved defence against neutrophils. Together, these data demonstrate that DNA is a target of host immune cells and several antibiotics, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.
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- 2020
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17. Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding.
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Furniss, R. Christopher D., Kaderabkova, Nikol, Barker, Declan, Bernal, Patricia, Maslova, Evgenia, Antwi, Amanda A. A., McNeil, Helen E., Pugh, Hannah L., Dortet, Laurent, Blair, Jessica M. A., Larrouy-Maumus, Gerald, McCarthy, Ronan R., Gonzalez, Diego, and Mavridou, Despoina A. I.
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- 2022
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18. Cytochrome c biogenesis System I
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Stevens, Julie M., Mavridou, Despoina A. I., Hamer, Rebecca, Kritsiligkou, Paraskevi, Goddard, Alan D., and Ferguson, Stuart J.
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- 2011
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19. Local frustration determines loop opening in protein-protein association
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Stelzl, Lukas S., Mavridou, Despoina A. I., Baldwin, Andrew J., Ferguson, Stuart J., Sansom, Mark S. P., and Redfield, Christina
- Abstract
Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic when others are rigid. More specifically, frustration is thought to play a key role in biomolecular recognition while it can also underpin the flexibility of binding sites. Here we show how a seemingly small chemical modification, the oxidation of two cysteine thiols to form a disulfide bond, during the biological function of the N-terminal domain of the bacterial oxidoreductase DsbD (nDsbD), introduces frustration. In oxidised nDsbD, local frustration disrupts the packing of the protective cap loop region against the active site of the protein allowing loop opening By contrast, in reduced nDsbD, lacking a disulfide bond, the cap loop is rigid, always shielding the active-site cysteines and protecting them from the otherwise oxidising environment of the bacterial periplasm. Our results point towards an intricate coupling between the dynamics of the active-site cysteines and those of the cap loop, which shapes the protein-protein association reactions of nDsbD resulting in optimised protein function.
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- 2019
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20. Harnessing the potential of bacterial oxidative folding to aid protein production.
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Slater, Sabrina L. and Mavridou, Despoina A.I.
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PROTEIN expression , *PROTEIN folding , *BACTERIAL proteins , *POST-translational modification , *RECOMBINANT proteins , *PROTEIN stability - Abstract
Protein folding is central to both biological function and recombinant protein production. In bacterial expression systems, which are easy to use and offer high protein yields, production of the protein of interest in its native fold can be hampered by the limitations of endogenous posttranslational modification systems. Disulfide bond formation, entailing the covalent linkage of proximal cysteine amino acids, is a fundamental posttranslational modification reaction that often underpins protein stability, especially in extracytoplasmic environments. When these bonds are not formed correctly, the yield and activity of the resultant protein are dramatically decreased. Although the mechanism of oxidative protein folding is well understood, unwanted or incorrect disulfide bond formation often presents a stumbling block for the expression of cysteine‐containing proteins in bacteria. It is therefore important to consider the biochemistry of prokaryotic disulfide bond formation systems in the context of protein production, in order to take advantage of the full potential of such pathways in biotechnology applications. Here, we provide a critical overview of the use of bacterial oxidative folding in protein production so far, and propose a practical decision‐making workflow for exploiting disulfide bond formation for the expression of any given protein of interest. [ABSTRACT FROM AUTHOR]
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- 2021
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21. Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.
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Sabnis, Akshay, Hagart, Katheryn L. H., Klöckner, Anna, Becce, Michele, Evans, Lindsay E., Furniss, R. Christopher D., Mavridou, Despoina A. I., Murphy, Ronan, Stevens, Molly M., Davies, Jane C., Larrouy-Maumus, Gérald J., Clarke, Thomas B., and Edwards, Andrew M.
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- 2021
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22. Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test
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Dortet, Laurent, Bonnin, Rémy, Pennisi, Ivana, Gauthier, Laura, Jousset, Agnès, Dabos, Laura, Furniss, R Christopher D, Mavridou, Despoina, Bogaerts, Pierre, Glupczynski, Youri, Potron, Anaïs, Plesiat, Patrick, Beyrouthy, Racha, Robin, Frédéric, Bonnet, Richard, Naas, Thierry, Filloux, Alain, Larrouy-Maumus, Gerald, Gauthier, Lauraine, AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Centre National de Référence de la Résistance aux Antibiotiques (CNR), Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), Cliniques Universitaires UCL de Mont-Godinne, Department of Food Science and Technology, Nestlé Research Center | Centre de recherche Nestlé [Lausanne], Nestlé S.A.-Nestlé S.A., Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte (M2iSH), Institut National de la Recherche Agronomique (INRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre de Recherche en Nutrition Humaine d'Auvergne (CRNH d'Auvergne), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Imperial College London, CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre Hospitalier Régional Universitaire [Besançon] (CHRU Besançon), Nestlé Research Center, Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte - Clermont Auvergne (M2iSH), Institut National de la Recherche Agronomique (INRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne (UCA)-Centre de Recherche en Nutrition Humaine d'Auvergne (CRNH d'Auvergne), Service de parasitologie - mycologie [CHU Pitié-Salpétrière], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Pitié-Salpêtrière [APHP], Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre de Recherche en Nutrition Humaine d'Auvergne (CRNH d'Auvergne)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), UCL - SSS/IREC/MONT - Pôle Mont Godinne, and UCL - (MGD) Laboratoire de biologie clinique
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0301 basic medicine ,Microbiology (medical) ,medicine.drug_class ,Polymyxin ,030106 microbiology ,Drug resistance ,Microbial Sensitivity Tests ,medicine.disease_cause ,Microbiology ,Bacterial genetics ,Lipid A ,03 medical and health sciences ,Drug Resistance, Bacterial ,medicine ,Escherichia coli ,Pharmacology (medical) ,Polymyxins ,Pharmacology ,biology ,Chemistry ,Escherichia coli Proteins ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Plasmid-mediated resistance ,Chromosomes, Bacterial ,biology.organism_classification ,Enterobacteriaceae ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,3. Good health ,Infectious Diseases ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,lipids (amino acids, peptides, and proteins) ,Bacteria ,Plasmids - Abstract
Background Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. Objectives To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. Methods We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in Results Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. Conclusions The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.
- Published
- 2018
23. The clue is in the lipid A: Rapid detection of colistin resistance.
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Furniss, R. Christopher D., Kostrzewa, Markus, Mavridou, Despoina A. I., and Larrouy-Maumus, Gerald
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COLISTIN ,LIPIDS ,POLYMYXIN B ,GRAM-negative bacterial diseases ,DRUG resistance in microorganisms ,TANDEM mass spectrometry - Abstract
L-Ara4N modification of the 4'-phosphate of native lipid A is characteristic of colistin-resistant E. coli strains carrying chromosomal mutations; this modification often co-occurs with PEtN modification of the 1-phosphate of the lipid A structure. For colistin-susceptible strains, only one major peak at I m/z i 1,796.2, corresponding to native lipid A, was detected, whereas for colistin-resistant strains, an additional peak at I m/z i 1,919.2 was present; the latter arises from the addition of a PEtN moiety to the 1-phosphate group of lipid A (Fig 1, left column, bottom panel). I coli i strains; colistin-susceptible strains had a PRR of zero, whereas colistin-resistant strains had a positive PRR. [Extracted from the article]
- Published
- 2020
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24. The heme auxotroph <italic>Caenorhabditis elegans</italic> can cleave the thioether bonds of <italic>c</italic>‐type cytochromes.
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Murphey, Annie C., Mavridou, Despoina A. I., Hodgkin, Jonathan, and Ferguson, Stuart J.
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HEME , *AUXOTROPHY , *CAENORHABDITIS elegans , *CYTOCHROMES , *POLYPEPTIDES - Abstract
Heme is essential and synthesized via highly regulated processes. For this reason, most organisms strive to recycle it or acquire it from their environment. When heme is bound to proteins noncovalently, degradation of the polypeptide is sufficient to release it. However, in some hemoproteins, such as
c ‐type cytochromes, heme is covalently bound to the protein backbone. We use the heme auxotrophCaenorhabditis elegans to investigate if cytochromesc can be a heme source, and we show that this organism must encode a novel system which specifically cleaves the thioether bonds ofc ‐type cytochromes. We also find that at limiting heme concentrations, while somatic tissues develop normally the germline fails to proliferate, suggesting the presence of a heme‐sensing checkpoint inC. elegans . [ABSTRACT FROM AUTHOR]- Published
- 2018
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25. The interplay between the disulfide bond formation pathway and cytochrome c maturation in Escherichia coli
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Mavridou, Despoina A.I., Ferguson, Stuart J., and Stevens, Julie M.
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- 2012
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26. The P aracoccus denitrificans Nar K-like nitrate and nitrite transporters-probing nitrate uptake and nitrate/nitrite exchange mechanisms.
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Goddard, Alan D., Bali, Shilpa, Mavridou, Despoina A.I., Luque‐Almagro, Victor M., Gates, Andrew J., Dolores Roldán, M., Newstead, Simon, Richardson, David J., and Ferguson, Stuart J.
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PARACOCCUS denitrificans ,BIOLOGICAL membranes ,PERIPLASM ,ESCHERICHIA coli ,STRUCTURAL models - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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27. The pUltra plasmid series: A robust and flexible tool for fluorescent labeling of Enterobacteria.
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Mavridou, Despoina A.I., Gonzalez, Diego, Clements, Abigail, and Foster, Kevin R.
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ENTEROBACTERIACEAE , *PLASMIDS , *ORGANISMS , *PROTEIN genetics , *FLOW cytometry - Abstract
Fluorescent labeling has been an invaluable tool for the study of living organisms and bacterial species are no exception to this. Here we present and characterize the pUltra plasmids 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 range of Enterobacteria. The amount of expressed fluorophore from these genetic constructs is such, that the contours of the cells can be delineated on the basis of the fluorescent signal only. In addition, labeling through the pUltra plasmids can be used successfully for fluorescence and confocal microscopy while unambiguous distinction of cells labeled with different colors can be carried out efficiently by microscopy or flow cytometry. We compare the labeling provided by the pUltra plasmids with that of another plasmid series encoding fluorescent proteins and we show that the pUltra constructs are vastly superior in signal intensity and discrimination power without having any detectable growth rate effects for the bacterial population. We also use the pUltra plasmids to produce mixtures of differentially labeled pathogenic Escherichia , Shigella and Salmonella species which we test during infection of mammalian cells. We find that even inside the host cell, different strains can be distinguished effortlessly based on their fluorescence. We, therefore, conclude that the pUltra plasmids are a powerful labeling tool especially useful for complex biological experiments such as the visualization of ecosystems of different bacterial species or of enteric pathogens in contact with their hosts. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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28. An Extended Active-site Motif Controls the Reactivity of the Thioredoxin Fold.
- Author
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Mavridou, Despoina A. I., Saridakis, Emmanuel, Kritsiligkou, Paraskevi, Mozley, Erin C., Ferguson, Stuart J., and Redfield, Christina
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THIOREDOXIN , *THIOREDOXIN-interacting protein , *PROTEIN folding , *C-terminal binding proteins , *OXIDATION-reduction reaction - Abstract
Proteins belonging to the thioredoxin (Trx) superfamily are abundant in all organisms. They share the same structural features, arranged in a seemingly simple fold, but they perform a multitude of functions in oxidative protein folding and electron transfer pathways. We use the C-terminal domain of the unique transmembrane reductant conductor DsbD as a model for an in-depth analysis of the factors controlling the reactivity of the Trx fold. We employ NMR spectroscopy, x-ray crystallography, mutagenesis, in vivo functional experiments applied to DsbD, and a comparative sequence analysis of Trx-fold proteins to determine the effect of residues in the vicinity of the active site on the ionization of the key nucleophilic cysteine of the -CXXC- motif. We show that the function and reactivity of Trx-fold proteins depend critically on the electrostatic features imposed by an extended active-site motif. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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29. Probing Heme Delivery Processes in Cytochrome c Biogenesis System I.
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Mavridou, Despoina A. I., Clark, Matthew N., Choulat, Cendie, Ferguson, Stuart J., and Stevens, Julie M.
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HEME , *CYTOCHROME c , *ORIGIN of life , *ESCHERICHIA coli , *QUINONE - Abstract
Cytochromes c comprise a diverse and widespread family of proteins containing covalently bound heme that are central to the life of most organisms. In many bacteria and in certain mitochondria, the synthesis of cytochromes c is performed by a complex post-translational modification apparatus called System I (or cytochrome c maturation, Ccm, system). In Escherichia coli, there are eight maturation proteins, several of which are involved in heme handling, but the mechanism of heme transfer from one protein to the next is not known. Attachment of the heme to the apocytochrome occurs via a novel covalent bond to a histidine residue of the heme chaperone CcmE. The discovery of a variant maturation system (System I*) has provided a new tool for studying cytochrome c assembly because the variant CcmE functions via a cysteine residue in the place of the histidine of System I. In this work, we use site-directed mutagenesis on both maturation systems to probe the function of the individual component proteins as well as their concerted action in transferring heme to the cytochrome c substrate. The roles of CcmA, CcmC, CcmE, and CcmF in the heme delivery process are compared between Systems I and I*. We show that a previously proposed quinone-binding site on CcmF is not essential for either system. Significant differences in the heme chemistry involved in the formation of cytochromes c in the variant system add new pieces to the cytochrome c biogenesis puzzle. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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30. Cytochrome c assembly.
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Mavridou, Despoina A. I., Ferguson, Stuart J., and Stevens, Julie M.
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CYTOCHROMES , *PROTEINS , *CHARGE exchange , *PHOTOSYNTHESIS , *HEME - Abstract
Cytochromes c are central proteins in energy transduction processes by virtue of their functions in electron transfer in respiration and photosynthesis. They have heme covalently attached to a characteristic CXXCH motif via protein-catalyzed post-translational modification reactions. Several systems with diverse constituent proteins have been identified in different organisms and are required to perform the heme attachment and associated functions. The necessary steps are translocation of the apocytochrome polypeptide to the site of heme attachment, transport and provision of heme to the appropriate compartment, reduction and chaperoning of the apocytochrome, and finally, formation of the thioether bonds between heme and two cysteines in the cytochrome. Here we summarize the established classical models for these processes and present recent progress in our understanding of the individual steps within the different cytochrome c biogenesis systems. © 2013 IUBMB Life, 65(3):209-216, 2013 [ABSTRACT FROM AUTHOR]
- Published
- 2013
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31. H, C and N resonance assignments for the oxidized and reduced states of the N-terminal domain of DsbD from Escherichia coli.
- Author
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Mavridou, Despoina, Stelzl, Lukas, Ferguson, Stuart, and Redfield, Christina
- Abstract
Viability and pathogenicity of Gram-negative bacteria is linked to the cytochrome c maturation and the oxidative protein folding systems in the periplasm. The transmembrane reductant conductor DsbD is a unique protein which provides the necessary reducing power to both systems through thiol-disulfide exchange reactions in a complex network of protein-protein interactions. The N-terminal domain of DsbD (nDsbD) is the delivery point of the reducing power originating from cytoplasmic thioredoxin to a variety of periplasmic partners. Here we report H, C and N assignments for resonances of nDsbD in its oxidized and reduced states. These assignments provide the starting point for detailed investigations of the interactions of nDsbD with its protein partners. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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32. Vital Dye Reaction and Granule Localization in Periplasm of Escherichia coli.
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Liyan Ping, Mavridou, Despoina A. I., Ferguson, Stuart J., Emberly, Eldon, and Westermann, Martin
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ESCHERICHIA coli , *FOOD poisoning , *COLIFORMS , *FOODBORNE diseases , *CELL proliferation - Abstract
Background: Tetrazolium salts are widely used in biology as indicators of metabolic activity - hence termed vital dyes - but their reduction site is still debated despite decades of intensive research. The prototype, 2,3,5- triphenyl tetrazolium chloride, which was first synthesized a century ago, often generates a single formazan granule at the old pole of Escherichia coli cells after reduction. So far, no explanation for their pole localization has been proposed. Method/Principal Findings: Here we provide evidence that the granules form in the periplasm of bacterial cells. A source of reducing power is deduced to be thiol groups destined to become disulfides, since deletion of dsbA, coding for thioloxidase, enhances the formation of reduced formazan. However, pervasive reduction did not result in a random distribution of formazan aggregates. In filamentous cells, large granules appear at regular intervals of about four normal cell-lengths, consistent with a diffusion-to-capture model. Computer simulations of a minimal biophysical model showed that the pole localization of granules is a spontaneous process, i.e. small granules in a normal size bacterium have lower energy at the poles. This biased their diffusion to the poles. They kept growing there and eventually became fixed. Conclusions: We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for reevaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts. The localization of formazan granules in E. coli cells can now be understood. In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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33. A Pivotal Heme-transfer Reaction Intermediate in Cytochrome c Biogenesis.
- Author
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Mavridou, Despoina A. I., Stevens, Julie M., Mönkemeyer, Leonie, Daltrop, Oliver, Gleria, Katalin di, Kessler, Benedikt M., Ferguson, Stuart J., and Allen, James W. A.
- Subjects
- *
CYTOCHROMES , *PROTEINS , *PHOTOSYNTHESIS , *RESPIRATION , *BACTERIA - Abstract
c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
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34. Oxidation State-dependent Protein-Protein Interactions in Disulfide Cascades.
- Author
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Mavridou, Despoina A. I., Saridakis, Emmanuel, Kritsiligkou, Paraskevi, Goddard, Alan D., Stevens, Julie M., Ferguson, Stuart J., and Redfield, Christina
- Subjects
- *
PROTEIN-protein interactions , *GRAM-negative bacteria , *EXCHANGE reactions , *MAGNETIC resonance microscopy , *X-ray crystallography - Abstract
Bacterial growth and pathogenicity depend on the correct formation of disulfide bonds, a process controlled by the Dsb system in the periplasm of Gram-negative bacteria. Proteins with a thioredoxin fold play a central role in this process. A general feature of thiol-disulfide exchange reactions is the need to avoid a long lived product complex between protein partners. We use a multidisciplinary approach, involving NMR, x-ray crystallography, surface plasmon resonance, mutagenesis, and in vivo experiments, to investigate the interaction between the two soluble domains of the transmembrane reductant conductor DsbD. Our results show oxidation state-dependent affinities between these two domains. These observations have implications for the interactions of the ubiquitous thioredoxin-like proteins with their substrates, provide insight into the key role played by a unique redox partner with an immunoglobulin fold, and are of general importance for oxidative protein-folding pathways in all organisms. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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- View/download PDF
35. Novel polycarboxylated EDTA-type cyclodextrins as ligands for lanthanide binding: study of their luminescence, relaxivity properties of Gd(III) complexes, and PM3 theoretical calculations.
- Author
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Maffeo, Davide, Lampropoulou, Maria, Fardis, Michael, Lazarou, Yannis G., Mavridis, Irene M., Mavridou, Despoina A. I., Urso, Elena, Pratsinis, Harris, Kletsas, Dimitris, and Yannakopoulou, Konstantina
- Published
- 2010
- Full Text
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36. Active-site Properties of the Oxidized and Reduced C-terminal Domain of DsbD Obtained by NMR Spectroscopy
- Author
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Mavridou, Despoina A.I., Stevens, Julie M., Ferguson, Stuart J., and Redfield, Christina
- Subjects
- *
NUCLEAR magnetic resonance , *HYDROGEN-ion concentration , *NUCLEAR magnetic resonance spectroscopy , *VOLUMETRIC analysis - Abstract
Abstract: The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteine residues in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of 13C, 15N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localized conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/oxidized transition. pK a values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side-chains of Asp455 and Glu468 in the active site contribute to the unusually high pK a value, 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/E468Q, the pK a value of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK a value of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2). [Copyright &y& Elsevier]
- Published
- 2007
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37. Bacteria Use Collective Behavior to Generate Diverse Combat Strategies.
- Author
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Mavridou, Despoina A.I., Gonzalez, Diego, Kim, Wook, West, Stuart A., and Foster, Kevin R.
- Subjects
- *
BACTERIA behavior , *COLLECTIVE behavior , *ESCHERICHIA coli , *COLICINS , *BACTERIAL colonies - Abstract
Summary Animals have evolved a wide diversity of aggressive behavior often based upon the careful monitoring of other individuals. Bacteria are also capable of aggression, with many species using toxins to kill or inhibit their competitors. Like animals, bacteria also have systems to monitor others during antagonistic encounters, but how this translates into behavior remains poorly understood. Here, we use colonies of Escherichia coli carrying colicin-encoding plasmids as a model for studying antagonistic behavior. We show that in the absence of threat, dispersed cells with low reproductive value produce colicin toxins spontaneously, generating efficient pre-emptive attacks. Cells can also respond conditionally to toxins released by clonemates via autoinduction or other genotypes via competition sensing. The strength of both pre-emptive and responsive attacks varies widely between strains. We demonstrate that this variability occurs easily through mutation by rationally engineering strains to recapitulate the diversity in naturally occurring strategies. Finally, we discover that strains that can detect both competitors and clonemates are capable of massive coordinated attacks on competing colonies. This collective behavior protects established colonies from competitors, mirroring the evolution of alarm calling in the animal world. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
38. The uronic acid content of coccolith-associated polysaccharides provides insight into coccolithogenesis and past climate.
- Author
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Lee, Renee B. Y., Mavridou, Despoina A. I., Papadakos, Grigorios, McClelland, Harry L. O., and Rickaby, Rosalind E. M.
- Published
- 2016
- Full Text
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39. Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces.
- Author
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Furniss, R. Christopher D., Wen Wen Low, Mavridou, Despoina A. I., Dagley, Laura F., Webb, Andrew I., Tate, Edward W., and Clements, Abigail
- Subjects
- *
ESCHERICHIA coli O157:H7 , *METALLOPROTEINASES , *CELL membranes , *PROTEOMICS , *CD55 antigen , *EPITHELIAL cells - Abstract
Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing lesion on the surface of infected cells causes significant remodeling of the host cell surface; however, limited information is available about changes at the protein level. Here we employed plasma membrane profiling, a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor cluster of differentiation 55 (CD55) exhibited the greatest reduction in cell-surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE and found that StcEmediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
40. Identification and characterisation of the type VI secretion system in Rhodobacter sphaeroides
- Author
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Potter, Kathryn Elizabeth, Mavridou, Despoina, and Foster, Kevin
- Abstract
The type VI secretion system (T6SS) is a versatile multi-component nanomachine found in 25% of Gram-negative bacteria. It translocates effector proteins into neighbouring eukaryotic or prokaryotic cells, and therefore it is important for bacterial virulence, as well as interbacterial competition. Although initially T6SSs were considered to be primarily contact-dependent weapons de- ployed during bacterial warfare, recently T6SSs that secrete effector proteins to sequester common goods from the environment have also been identified. Characterised examples of such systems play a role in the uptake of zinc, manganese, iron, copper and molybdate ions. The mechanism behind T6SS- dependent metal ion acquisition involves secretion of a metal-binding protein which sequesters metal ions from the extracellular milieu. The subsequent steps by which cells uptake the sequestered metal ions remain largely elusive, nonetheless it it is known that, in some cases, interaction of the secreted protein with a TonB-dependent outer membrane transporter is required. Here we report the previously uncharacterised T6SS of Rhodobacter sphaeroides WS8N. By performing bioinformatic analyses on the putative T6SS cluster we find that it encodes all 13 core T6SS proteins along with four proteins known to be involved in T6SS regulation (TagE-H), the T6SS tip sharpening protein PAAR, the peptidoglycan-binding protein TagL and an additional seven hypothetical proteins. We also find the presence of a T6SS to be strain, and not species, specific in the Rhodobacteraceae family of proteobacteria. Further, we investigated the conditions under which the the T6SS of R. sphaeroides WS8N is active and demonstrate that, its function is required for photoheterotrophic growth under iron-limited conditions. In particular, presence of this T6SS allows growth of R. sphaeroides WS8N in limited amounts of both Fe²⁺ and Fe³⁺, but does not support growth in iron-depleted media supplemented with heme. Finally, we performed preliminary biochemical studies of the C-terminal extension of VgrG, which showed the potential to bind iron. This suggests that VgrG might be the secreted protein facilitating the T6SS-dependent uptake of iron. The data presented in this study represents the first characterisation of a T6SS from the Rhodobacteraceae family of proteobacteria, informing us on the role of this system in a class of organisms that heavily depend on the use of iron for their growth.
- Published
- 2021
41. The role of the DSB system in antimicrobial resistance
- Author
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Kaderabkova, Nikol, Mavridou, Despoina, and Filloux, Alain
- Abstract
Extensive use of antibiotics in medicine and agriculture has led to increasing emergence of antimicrobial resistance in bacterial populations. Dwindling resources in the discovery of novel active compound leads and the increasing demands for safety and efficacy of new drugs mean that we are now faced with treatment failures due to multi-drug resistant pathogens. In the quest for new targets that will enable us to counter antibiotic resistance, it is often ignored that many resistance mechanisms precede the clinical use of antibiotics. Instead, the ability to adapt, survive and bypass the toxicity of many chemical compounds is wired within the bacterial genome. Continuous inter-strain and inter-species competition have given microorganisms tools to thrive under conditions of chemical warfare. Recognising this is important when characterising mechanisms underpinning bacterial antimicrobial resistance, as it can lead to novel strategies that can help us bypass it. The work described here explores the connection between the disulfide bond formation system, a key oxidative protein folding pathway in the cell envelope of Gram-negative bacteria, and two widespread antimicrobial resistance mechanisms, b-lactamase catalysed hydrolysis of b-lactam antibiotics and efflux-mediated drug expulsion. It is demonstrated that oxidative-protein-folding-mediated proteostasis is crucial for both resistance mechanisms, and its inhibition can sensitise multidrug-resistant pathogens to existing antibiotics. Preliminary results from an experimental evolution approach, set the scene for future exploration of the importance of disulfide linkages for the capacity of b-lactamase enzymes to evolve under selective pressure. Together, these findings aim to address the mechanistic basis of a new avenue for antibiotic adjuvant therapy, whereby targeting a non-essential process would allow us to potentiate existing antibiotics towards previously resistant bacterial strains. With novel essential targets against bacteria being scarce, adjuvant approaches like this one could prolong the use and efficacy of existing drugs against some of the most resistant Gram-negative pathogens.
- Published
- 2021
- Full Text
- View/download PDF
42. Lifestyle transitions and adaptive pathogenesis of Pseudomonas aeruginosa.
- Author
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Valentini, Martina, Gonzalez, Diego, Mavridou, Despoina AI, and Filloux, Alain
- Subjects
- *
PSEUDOMONAS aeruginosa , *VIRULENCE of bacteria , *BIOFILMS , *BACTERIAL cells , *BACTERIAL communities , *CELL-mediated cytotoxicity - Abstract
Pseudomonas aeruginosa acute and chronic infections are of great concern to human health, especially in hospital settings. It is currently assumed that P. aeruginosa has two antagonistic pathogenic strategies that parallel two different lifestyles; free-living cells are predominantly cytotoxic and induce an acute inflammatory reaction, while biofilm-forming communities cause refractory chronic infections. Recent findings suggest that the planktonic-to-sessile transition is a complex, reversible and overall dynamic differentiation process. Here, we examine how the Gac/Rsm regulatory cascade, a key player in this lifestyle switch, endows P. aeruginosa with both a permissive lifecycle in nature and flexible virulence strategy during infection. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
43. Designing and identifying β-hairpin peptide macrocycles with antibiotic potential.
- Author
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Randall, Justin R., DuPai, Cory D., Cole, T. Jeffrey, Davidson, Gillian, Groover, Kyra E., Slater, Sabrina L., Mavridou, Despoina A. I., Wilke, Claus O., and Davies, Bryan W.
- Subjects
- *
PEPTIDE antibiotics , *ANTIMICROBIAL peptides , *ANTIBIOTICS - Abstract
The article presents a study which explores about the designing and identifying beta-hairpin peptide macrocycles with antibiotic potential. It mentions that findings of the study provide a synthetic strategy for structured macrocyclic peptide design and discovery while also elucidating characteristics important for beta-hairpin antimicrobial peptide activity.
- Published
- 2023
- Full Text
- View/download PDF
44. Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells
- Author
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Maria A Sainz-Polo, Julian Parkhill, Amy K. Cain, Laura M. Nolan, Alain Filloux, Despoina A. I. Mavridou, Gordon Dougan, David Albesa-Jové, R. Christopher D. Furniss, Thomas Clamens, Eleni Manoli, Medical Research Council (UK), European Commission, Biotechnology and Biological Sciences Research Council (UK), Ministerio de Economía y Competitividad (España), Fundación Biofísica Bizkaia, Eusko Jaurlaritza, Cain, Amy K., Furniss, R. Christopher D., Albesa-Jové, David, Parkhill, Julian, Mavridou, Despoina A. I., Filloux, Alain, Imperial College London, Cain, Amy K [0000-0002-4230-6572], Furniss, R Christopher D [0000-0002-5806-5099], Albesa-Jové, David [0000-0003-2904-8203], Parkhill, Julian [0000-0002-7069-5958], Mavridou, Despoina A I [0000-0002-7449-1151], Filloux, Alain [0000-0003-1307-0289], Apollo - University of Cambridge Repository, and Mavridou, Despoina AI [0000-0002-7449-1151]
- Subjects
Microbiology (medical) ,MECHANISM ,Multiprotein complex ,Immunology ,Bacterial Toxins ,EFFECTOR ,Plasma protein binding ,LYS CATALYTIC TRIAD ,Applied Microbiology and Biotechnology ,Microbiology ,Article ,DELIVERY ,AMIDASE ,Bacterial Proteins ,1108 Medical Microbiology ,Bacterial genetics ,REVEALS ,Genetics ,Protein biosynthesis ,Secretion ,Asparagine ,Type VI secretion system ,Science & Technology ,biology ,HYDROLASE ,Effector ,Chemistry ,Bacteriology ,Cell Biology ,Type VI Secretion Systems ,biology.organism_classification ,GENE ,Biochemistry ,Multiprotein Complexes ,Protein Biosynthesis ,Pseudomonas aeruginosa ,Life Sciences & Biomedicine ,Bacteria ,0605 Microbiology ,Protein Binding - Abstract
The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria., L.M.N. was supported by Medical Research Council (MRC) Grant MR/N023250/1 and a Marie Curie Fellowship (PIIF-GA-2013-625318). A.F. was supported by MRC Grants MR/K001930/1 and MR/N023250/1 and Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/N002539/1. R.C.D.F. and D.A.I.M. were supported by the MRC Career Development Award MR/M009505/1. D.A.-J. acknowledges support by the MINECO Contract CTQ2016-76941-R, Fundación Biofísica Bizkaia, the Basque Excellence Research Centre (BERC) program and IT709-13 of the Basque Government, and Fundación BBVA. M.A.S.-P. was supported by the MINECO under the “Juan de la Cierva Postdoctoral program” (position FJCI-2015-25725).
- Published
- 2021
45. The CcmC-CcmE interaction during cytochrome c maturation by System I is driven by protein-protein and not protein- heme contacts.
- Author
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Shevket, Shevket H., Gonzalez, Diego, Cartwright, Jared L., Kleanthous, Colin, Ferguson, Stuart J., Redfield, Christina, and Mavridou, Despoina A. I.
- Subjects
- *
CYTOCHROME c , *GRAM-negative bacteria , *PROTEIN-protein interactions , *HEME , *PLANT mitochondria - Abstract
Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA-I) called System I. Despite decades of study, mechanistic understanding of the protein-protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp47, Gln50, and Arg55 on CcmC; Arg73, Asp101, and Glu105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli. Moreover, employingNMRspectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein-protein rather than protein-heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
46. c-Type Cytochrome Biogenesis Can Occur via a Natural Ccm System Lacking CcmH, CcmG, and the Heme-binding Histidine of CcmE.
- Author
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Goddard, Alan D., Stevens, Julie M., Feng Rao, Mavridou, Despoina A. I., Weelee Chan, Richardson, David J., Allen, James W. A., and Ferguson, Stuart J.
- Subjects
- *
CYTOCHROME c , *MITOCHONDRIA formation , *HEME , *HISTIDINE , *DESULFOVIBRIO - Abstract
The Ccm cytochrome c maturation System I catalyzes covalent attachment of heme to apocytochromes c in many bacterial species and some mitochondria. A covalent, but transient, bond between heme and a conserved histidine in CcmE along with an interaction between CcmH and the apocytochrome have been previously indicated as core aspects of the Ccm system. Here, we show that in the Ccm system from Desulfovibrio desulfuricans, no CcmH is required, and the holo-CcmE covalent bond occurs via a cysteine residue. These observations call for reconsideration of the accepted models of System I-mediated c-type cytochrome biogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
47. Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis.
- Author
-
Kadeřábková N, Furniss RCD, Maslova E, Eisaiankhongi L, Bernal P, Filloux A, Landeta C, Gonzalez D, McCarthy RR, and Mavridou DAI
- Abstract
Critical Gram-negative pathogens, like Pseudomonas , Stenotrophomonas and Burkholderia , have become resistant to most antibiotics. Complex resistance profiles together with synergistic interactions between these organisms increase the likelihood of treatment failure in distinct infection settings, for example in the lungs of cystic fibrosis patients. Here, we discover that cell envelope protein homeostasis pathways underpin both antibiotic resistance and cross-protection in CF-associated bacteria. We find that inhibition of oxidative protein folding inactivates multiple species-specific resistance proteins. Using this strategy, we sensitize multi-drug resistant Pseudomonas aeruginosa to β-lactam antibiotics and demonstrate promise of new treatment avenues for the recalcitrant pathogen Stenotrophomonas maltophilia . The same approach also inhibits cross-protection between resistant S. maltophilia and susceptible P. aeruginosa , allowing eradication of both commonly co-occurring CF-associated organisms. Our results provide the basis for the development of next-generation strategies that target antibiotic resistance, while also impairing specific interbacterial interactions that enhance the severity of polymicrobial infections., Competing Interests: DECLARATION OF INTERESTS: The authors declare no competing interests.
- Published
- 2023
- Full Text
- View/download PDF
48. The biogenesis of β-lactamase enzymes.
- Author
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Kaderabkova N, Bharathwaj M, Furniss RCD, Gonzalez D, Palmer T, and Mavridou DAI
- Subjects
- Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, beta-Lactamase Inhibitors, Penicillins, beta-Lactamases genetics, beta-Lactamases metabolism
- Abstract
The discovery of penicillin by Alexander Fleming marked a new era for modern medicine, allowing not only the treatment of infectious diseases, but also the safe performance of life-saving interventions, like surgery and chemotherapy. Unfortunately, resistance against penicillin, as well as more complex β-lactam antibiotics, has rapidly emerged since the introduction of these drugs in the clinic, and is largely driven by a single type of extra-cytoplasmic proteins, hydrolytic enzymes called β-lactamases. While the structures, biochemistry and epidemiology of these resistance determinants have been extensively characterized, their biogenesis, a complex process including multiple steps and involving several fundamental biochemical pathways, is rarely discussed. In this review, we provide a comprehensive overview of the journey of β-lactamases, from the moment they exit the ribosomal channel until they reach their final cellular destination as folded and active enzymes.
- Published
- 2022
- Full Text
- View/download PDF
49. Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic.
- Author
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Humphrey M, Larrouy-Maumus GJ, Furniss RCD, Mavridou DAI, Sabnis A, and Edwards AM
- Subjects
- Anti-Bacterial Agents pharmacology, Drug Resistance, Bacterial genetics, Escherichia coli genetics, Microbial Sensitivity Tests, Plasmids, Polymyxins, Colistin pharmacology, Escherichia coli Proteins genetics
- Abstract
Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug-resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to bacterial death and lysis. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilized colistin-resistance ( mcr ) genes. Both these colistin-resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilization of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilization of the outer membrane of colistin-resistant isolates by the polymyxin is in turn sufficient to sensitize bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in these E. coli isolates is due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.
- Published
- 2021
- Full Text
- View/download PDF
50. The Breadth and Molecular Basis of Hcp-Driven Type VI Secretion System Effector Delivery.
- Author
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Howard SA, Furniss RCD, Bonini D, Amin H, Paracuellos P, Zlotkin D, Costa TRD, Levy A, Mavridou DAI, and Filloux A
- Subjects
- Biological Transport, Escherichia coli metabolism, Pseudomonas aeruginosa chemistry, Type VI Secretion Systems classification, Bacterial Proteins genetics, Bacterial Proteins metabolism, Pseudomonas aeruginosa genetics, Type VI Secretion Systems genetics, Type VI Secretion Systems metabolism
- Abstract
The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2-T6SS, and H3-T6SS), to perform in vivo pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coli. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function. IMPORTANCE The type VI secretion system (T6SS) is an effective weapon used by bacteria to outgrow or kill competitors. It can be used by endogenous commensal microbiota to prevent invasion by pathogens or by pathogens to overcome resident flora and successfully colonize a host or a specific environmental niche. The T6SS is a key contributor to this continuous arms race between organisms as it delivers a multitude of toxins directed at essential processes, such as nucleic acid synthesis and replication, cell wall and membrane integrity, protein synthesis, or cofactor abundance. Many T6SS toxins with unknown function remain to be discovered, whose yet-uncharacterized targets could be exploited for antimicrobial drug design. The systematic search for these toxins is not facilitated by the presence of readily recognizable T6SS motifs, and unbiased screening approaches are thus required. Here, we successfully used a known shuttle for cargo T6SS effectors, Hcp, as bait to identify uncharacterized toxins.
- Published
- 2021
- Full Text
- View/download PDF
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