48 results on '"Mulholland F"'
Search Results
2. Glycolytic enzymes associated with the cell surface of Streptococcus pneumoniae are antigenic in humans and elicit protective immune responses in the mouse
- Author
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LING, E., FELDMAN, G., PORTNOI, M., DAGAN, R., OVERWEG, K., MULHOLLAND, F., CHALIFA-CASPI, V., WELLS, J., and MIZRACHI-NEBENZAHL, Y.
- Published
- 2004
3. A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk
- Author
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I'Anson, K.J.A., Movahedi, S., Griffin, H.G., Gasson, M.J., and Mulholland, F.
- Subjects
Bacteria -- Growth ,Aminopeptidases -- Research ,Amino acid sequence -- Analysis ,Biological sciences - Abstract
Glutamyl aminopeptidase (PepA) produces optimal growth of the bacteria Lactococcus lactis MG1363 in milk. However, the aminopeptidase is not essential for bacterial growth. Mutants lacking PepA show normal cell density but growth during the exponential phases is slower. Escherichia coli cells expressing the pepA gene produce active PepA. The complete open reading frame (ORF) of the pepA gene encodes a protein whose N-terminal is similar to PepA isolated from the bacteria. There are no N-terminal signal sequences or hydrophobicity sites in pepA.
- Published
- 1995
4. Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability
- Author
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Guccione, E.J., Kendall, J.J., Hitchcock, A., Garg, N., White, M.A., Mulholland, F., Poole, R.K., and Kelly, D.J.
- Abstract
Campylobacter jejuni, the most frequent cause of food-borne bacterial gastroenteritis worldwide, is a microaerophile that has to survive high environmental oxygen tensions, adapt to oxygen limitation in the intestine and resist host oxidative attack. Here, oxygen-dependent changes in C. jejuni physiology were studied at constant growth rate using carbon (serine)-limited continuous chemostat cultures. We show that a perceived aerobiosis scale can be calibrated by the acetate excretion flux, which becomes zero when metabolism is fully aerobic (100% aerobiosis). Transcriptome changes in a downshift experiment from 150% to 40% aerobiosis revealed many novel oxygen-regulated genes and highlighted re-modelling of the electron transport chains. A label-free proteomic analysis showed that at 40% aerobiosis, many proteins involved in host colonisation (e.g. PorA, CadF, FlpA, CjkT) became more abundant. PorA abundance increased steeply below 100% aerobiosis. In contrast, several citric-acid cycle enzymes, the peptide transporter CstA, PEB1 aspartate/glutamate transporter, LutABC lactate dehydrogenase and PutA proline dehydrogenase became more abundant with increasing aerobiosis. We also observed a co-ordinated response of oxidative stress protection enzymes and Fe-S cluster biogenesis proteins above 100% aerobiosis. Our approaches reveal key virulence factors that respond to restricted oxygen availability and specific transporters and catabolic pathways activated with increasing aerobiosis. This article is protected by copyright. All rights reserved.
- Published
- 2017
5. RANDOM NOTES ON BRITISH FORESTRY BY A CANADIAN
- Author
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MULHOLLAND, F. D.
- Published
- 1938
6. THE PROGRESS OF FORESTRY IN BRITISH COLUMBIA
- Author
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Mulholland, F. D.
- Published
- 1936
7. Transcriptomics and proteomics show that selenium affects inflammation, ctoskeleton, and cancer pathways in human rectal biopsies
- Author
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Meplan, C., Johnson, I. T., Polley, A. C. J., Cockell, S., Bradburn, D. M., Commane, Danny M., Arasaradnam, R. P., Mulholland, F., Zupanic, A., Mathers, J. C., and Hesketh, J.
- Abstract
Epidemiologic studies highlight the potential role of dietary selenium (Se) in colorectal cancer prevention. Our goal was to elucidate whether expression of factors crucial for colorectal homoeostasis is affected by physiologic differences in Se status. Using transcriptomics and proteomics followed by pathway analysis, we identified pathways affected by Se status in rectal biopsies from 22 healthy adults, including 11 controls with optimal status (mean plasma Se = 1.43 μM) and 11 subjects with suboptimal status (mean plasma Se = 0.86 μM). We observed that 254 genes and 26 proteins implicated in cancer (80%), immune function and inflammatory response (40%), cell growth and proliferation (70%), cellular movement, and cell death (50%) were differentially expressed between the 2 groups. Expression of 69 genes, including selenoproteins W1 and K, which are genes involved in cytoskeleton remodelling and transcription factor NFκB signaling, correlated significantly with Se status. Integrating proteomics and transcriptomics datasets revealed reduced inflammatory and immune responses and cytoskeleton remodelling in the suboptimal Se status group. This is the first study combining omics technologies to describe the impact of differences in Se status on colorectal expression patterns, revealing that suboptimal Se status could alter inflammatory signaling and cytoskeleton in human rectal mucosa and so influence cancer risk.
- Published
- 2016
8. The NuGO proof of principle study package: a collaborative research effort of the European Nutrigenomics Organisation
- Author
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Baccini, M, Bachmaier, EM, Biggeri, A, Boekschoten, MV, Bouwman, FG, Brennan, L, Caesar, R, Cinti, S, Coort, SL, Crosley, K, Daniel, H, Drevon, CA, Duthie, S, Eijssen, L, Elliott, RM, van Erk, M, Evelo, C, Gibney, M, Heim, C, Horgan, GW, Johnson, IT, Kelder, T, Kleemann, R, Kooistra, T, van Iersel, MP, Mariman, EC, Mayer, C, McLoughlin, G, Müller, M, Mulholland, F, van Ommen, B, Polley, AC, Pujos-Guillot, E, Rubio-Aliaga, I, Roche, HM, de Roos, B, Sailer, M, Tonini, G, Williams, LM, de Wit, N, For the NuGO PPS Team, Università degli Studi di Firenze = University of Florence (UniFI), University of Aberdeen, Division of Human Nutrition, Wageningen University and Research [Wageningen] (WUR), Top Institute Food and Nutrition (TIFN), Maastricht University [Maastricht], School of Agriculture, Food Science and Veterinary Medicine, University College Dublin [Dublin] (UCD), University of Oslo (UiO), Università Politecnica delle Marche, Partenaires INRAE, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Institute of Food Research [Norwich], Biotechnology and Biological Sciences Research Council (BBSRC), Department of Physiological Genomics, Munich University, University College Dublin (UCD), Netherlands Organisation for Applied Scientific Research, Unité de Nutrition Humaine (UNH), Institut National de la Recherche Agronomique (INRA)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Clermont Université, TNO Kwaliteit van Leven, Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI), Humane Biologie, Bioinformatica, RS: CARIM School for Cardiovascular Diseases, and RS: NUTRIM - R4 - Gene-environment interaction
- Subjects
Endocrinology, Diabetes and Metabolism ,[SDV]Life Sciences [q-bio] ,MEDLINE ,030209 endocrinology & metabolism ,Physiological Sciences ,Bioinformatics ,03 medical and health sciences ,Voeding, Metabolisme en Genomica ,0302 clinical medicine ,Voeding ,Genetics ,Network of excellence ,Medicine ,030304 developmental biology ,Nutrition ,VLAG ,0303 health sciences ,business.industry ,Metabolism and Genomics ,Nutrigenomics ,immune-system ,Proof of concept ,Metabolisme en Genomica ,Commentary ,Engineering ethics ,Nutrition, Metabolism and Genomics ,business - Abstract
Acknowledgments This project is funded by the Nutrigenomics Organisation, EC funded Network of Excellence, grant nr.FOOD- 2004-506360.
- Published
- 2008
9. Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism
- Author
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Elliott, R.M., Elliott, R.M., de Roos, B., Duthie, S.J., Bouwman, F.G., Rubio Aliaga, I., Crosley, L.K., Mayer, C., Polley, A.C., Heim, C., Coort, S., Evelo, C.T., Mulholland, F., Daniel, H., Mariman, E.C., Johnson, I.T., Elliott, R.M., Elliott, R.M., de Roos, B., Duthie, S.J., Bouwman, F.G., Rubio Aliaga, I., Crosley, L.K., Mayer, C., Polley, A.C., Heim, C., Coort, S., Evelo, C.T., Mulholland, F., Daniel, H., Mariman, E.C., and Johnson, I.T.
- Abstract
There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.
- Published
- 2014
10. In vitro digestibility of β-casein and β-lactoglobulin under simulated human gastric and duodenal conditions: A multi-laboratory evaluation
- Author
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Mandalari, G. Adel-Patient, K. Barkholt, V. Baro, C. Bennett, L. Bublin, M. Gaier, S. Graser, G. Ladics, G.S. Mierzejewska, D. Vassilopoulou, E. Vissers, Y.M. Zuidmeer, L. Rigby, N.M. Salt, L.J. Defernez, M. Mulholland, F. Mackie, A.R. Wickham, M.S.J. Mills, E.N.C.
- Abstract
Initially the resistance to digestion of two cow's milk allergens, β-casein, and β-lactoglobulin (β-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both β-casein and β-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of β-casein digestion in the low-protease assay were slower, β-Lg being pepsin resistant. During duodenal digestion, β-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins. © 2009 Elsevier Inc.
- Published
- 2009
11. Inducible expression of the essential response regulator YycF in S.pneumoniae modulates expression of genes involved in fatty acid biosynthesis and affects membrane composition
- Author
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Mohedano, M.L., Overweg, K., de la Fuente, A., Reuter, M., Mulholland, F., Lopez, P., Wells, J.M., and SILS Other Research (FNWI)
- Published
- 2005
12. Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism
- Author
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Elliott, R. M., primary, de Roos, B., additional, Duthie, S. J., additional, Bouwman, F. G., additional, Rubio-Aliaga, I., additional, Crosley, L. K., additional, Mayer, C., additional, Polley, A. C., additional, Heim, C., additional, Coort, S. L., additional, Evelo, C. T., additional, Mulholland, F., additional, Daniel, H., additional, Mariman, E. C., additional, and Johnson, I. T., additional
- Published
- 2014
- Full Text
- View/download PDF
13. Metabolic Shift of Escherichia coli under Salt Stress in the Presence of Glycine Betaine
- Author
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Metris, A., primary, George, S. M., additional, Mulholland, F., additional, Carter, A. T., additional, and Baranyi, J., additional
- Published
- 2014
- Full Text
- View/download PDF
14. 2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting
- Author
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Bouwman, F.G., Bouwman, F.G., de Roos, B., Rubio Aliaga, I., Crosley, L.K., Duthie, S.J., Mayer, C., Horgan, G., Polley, A.C., Heim, C., Coort, S.L.M., Evelo, C.T.A., Mulholland, F., Johnson, I.T., Elliott, R.M., Daniel, H., Mariman, E.C., Bouwman, F.G., Bouwman, F.G., de Roos, B., Rubio Aliaga, I., Crosley, L.K., Duthie, S.J., Mayer, C., Horgan, G., Polley, A.C., Heim, C., Coort, S.L.M., Evelo, C.T.A., Mulholland, F., Johnson, I.T., Elliott, R.M., Daniel, H., and Mariman, E.C.
- Abstract
BACKGROUND: Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation. METHODS: Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach (multiplex immunoassay) were applied to investigate how fasting for 36h, as compared to 12h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva collected from ten healthy volunteers. RESULTS: Between-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF) analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells. CONCLUSIONS: Identification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics.
- Published
- 2011
15. The NuGO proof of principle study package: a collaborative research effort of the European Nutrigenomics Oganisation
- Author
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Baccini, M., Bachmaier, E.M., Biggeri, A., Boekschoten, M.V., Bouwman, F.G., Brennan, L., Caesar, R., Cinti, S., Coort, S.L., Crosley, K., Daniel, H., Drevon, C.A., Duthie, S., Eijssen, L., Elliott, R., van Erk, M.J., Evelo, C., Gibney, M.J., Heim, C., Horgan, G., Johnson, I.T., Kelder, T., Kleemann, R., Kooistra, T., van Iersel, M.P., Mariman, E.C.M., Mayer, C., McLoughlin, G., Müller, M.R., Mulholland, F., van Ommen, B., Polley, A.C., Pujos-Guillot, E., Rubio-Aliaga, I., Roche, H., de Roos, B., Sailer, M., Tonini, G., Williams, L.M., de Wit, N.J.W., Baccini, M., Bachmaier, E.M., Biggeri, A., Boekschoten, M.V., Bouwman, F.G., Brennan, L., Caesar, R., Cinti, S., Coort, S.L., Crosley, K., Daniel, H., Drevon, C.A., Duthie, S., Eijssen, L., Elliott, R., van Erk, M.J., Evelo, C., Gibney, M.J., Heim, C., Horgan, G., Johnson, I.T., Kelder, T., Kleemann, R., Kooistra, T., van Iersel, M.P., Mariman, E.C.M., Mayer, C., McLoughlin, G., Müller, M.R., Mulholland, F., van Ommen, B., Polley, A.C., Pujos-Guillot, E., Rubio-Aliaga, I., Roche, H., de Roos, B., Sailer, M., Tonini, G., Williams, L.M., and de Wit, N.J.W.
- Published
- 2008
16. Riboflavin Biosynthesis Is Associated with Assimilatory Ferric Reduction and Iron Acquisition by Campylobacter Jejuni.
- Author
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Gaskin, D.J.H., Holmes, K., Mulholland, F., Wells, J., Gaskin, D.J.H., Holmes, K., Mulholland, F., and Wells, J.
- Abstract
One of the pathways involved in the acquisition of the essential metal iron by bacteria involves the reduction of insoluble Fe3+ to soluble Fe2+, followed by transport of Fe2+ to the cytoplasm. Flavins have been implicated as electron donors in this poorly understood process. Ferrous iron uptake is essential for intestinal colonization by the important pathogen Campylobacter jejuni and may be of particular importance under low-oxygen conditions. In this study, the links among riboflavin biosynthesis, ferric reduction, and iron acquisition in C. jejuni NCTC11168 have been investigated. A riboflavin auxotroph was generated by inactivation of the ribB riboflavin biosynthesis gene (Cj0572), and the resulting isogenic ribB mutant only grew in the presence of exogenous riboflavin or the riboflavin precursor diacetyl but not in the presence of the downstream products flavin adenine dinucleotide and flavin mononucleotide. Riboflavin uptake was unaffected in the ribB mutant under iron-limited conditions but was lower in both the wild-type strain and the ribB mutant under iron-replete conditions. Mutation of the fur gene, which encodes an iron uptake regulator of C. jejuni, resulted in an increase in riboflavin uptake which was independent of the iron content of the medium, suggesting a role for Fur in the regulation of the as-yet-unknown riboflavin transport system. Finally, ferric reduction activity was independent of iron availability in the growth medium but was lowered in the ribB mutant compared to the wild-type strain and, conversely, increased in the fur mutant. Taken together, the findings confirm close relationships among iron acquisition, riboflavin production, and riboflavin uptake in C. jejuni
- Published
- 2007
17. Significance of low molecular weight nitrogen constituents in cheese milk and proteinase-negative variants of a Lactococcus lactis susp. cremoris strain on the production and maturation of Danbo cheese
- Author
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Sørensen, N. K., Qvist, K. B., Mulholland, F., Sørensen, N. K., Qvist, K. B., and Mulholland, F.
- Published
- 1996
18. Glycolytic enzymes associated with the cell surface ofStreptococcus pneumoniaeare antigenic in humans and elicit protective immune responses in the mouse.
- Author
-
LING, E., FELDMAN, G., PORTNOI, M., DAGAN, R., OVERWEG, K., MULHOLLAND, F., CHALIFA-CASPI, V., WELLS, J., and MIZRACHI-NEBENZAHL, Y.
- Subjects
STREPTOCOCCUS pneumoniae ,PNEUMOCOCCAL vaccines ,LUNG diseases ,NEISSERIA meningitidis ,IMMUNE response ,GEL electrophoresis ,HEAT shock proteins - Abstract
Streptococcus pneumoniaeis a leading cause of otitis media, sinusitis, pneumonia, bacteraemia and meningitis worldwide. The drawbacks associated with the limited number of various capsular polysaccharides that can be included in the polysaccharide-based vaccines focuses much attention on pneumococcal proteins as vaccine candidates. We extracted an enriched cell wall fraction fromS. pneumoniaeWU2. Approximately 150 soluble proteins could be identified by 2D gel electrophoresis. The proteins were screened by 2D-Western blotting using sera that were obtained longitudinally from children attending day-care centres at 18, 30 and 42 months of age and sera from healthy adult volunteers. The proteins were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Seventeen proteins were antigenic in children and adults, of which 13 showed an increasing antibody response with age in all eight children analysed. Two immunogenic proteins, fructose–bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a control protein with known low immunogenicity, heat shock protein 70 (DnaK), were expressed inEscherichia coli, purified and used to immunize mice. Mouse antibodies elicited to the recombinant (r) FBA and rGAPDH were cross-reactive with several genetically unrelated strains of different serotypes and conferred protection to respiratory challenge with virulent pneumococci. In addition, the FBA used in this study (NP_345117) does not have a human ortholog and warrants further investigation as a candidate for a pneumococcal vaccine. In conclusion, the immunoproteomics based approach utilized in the present study appears to be a suitable tool for identification of novelS. pneumoniaevaccine candidates. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
19. Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.
- Author
-
Elliott, R. M. (R. M.), de Roos, B. (B.), Duthie, S. J. (S. J.), Bouwman, F. G. (F. G.), Rubio-Aliaga, I. (I.), Crosley, L. K. (L. K.), Mayer, C. (C.), Polley, A. C. (A. C.), Heim, C. (C.), Coort, S. L. (S. L.), Evelo, C. T. (C. T.), Mulholland, F. (F.), Daniel, H. (H.), Mariman, E. C. (E. C.), Johnson, I. T. (I. T.), Elliott, R. M. (R. M.), de Roos, B. (B.), Duthie, S. J. (S. J.), Bouwman, F. G. (F. G.), Rubio-Aliaga, I. (I.), Crosley, L. K. (L. K.), Mayer, C. (C.), Polley, A. C. (A. C.), Heim, C. (C.), Coort, S. L. (S. L.), Evelo, C. T. (C. T.), Mulholland, F. (F.), Daniel, H. (H.), Mariman, E. C. (E. C.), and Johnson, I. T. (I. T.)
- Abstract
There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.
20. Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.
- Author
-
Elliott, R. M. (R. M.), de Roos, B. (B.), Duthie, S. J. (S. J.), Bouwman, F. G. (F. G.), Rubio-Aliaga, I. (I.), Crosley, L. K. (L. K.), Mayer, C. (C.), Polley, A. C. (A. C.), Heim, C. (C.), Coort, S. L. (S. L.), Evelo, C. T. (C. T.), Mulholland, F. (F.), Daniel, H. (H.), Mariman, E. C. (E. C.), Johnson, I. T. (I. T.), Elliott, R. M. (R. M.), de Roos, B. (B.), Duthie, S. J. (S. J.), Bouwman, F. G. (F. G.), Rubio-Aliaga, I. (I.), Crosley, L. K. (L. K.), Mayer, C. (C.), Polley, A. C. (A. C.), Heim, C. (C.), Coort, S. L. (S. L.), Evelo, C. T. (C. T.), Mulholland, F. (F.), Daniel, H. (H.), Mariman, E. C. (E. C.), and Johnson, I. T. (I. T.)
- Abstract
There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.
21. 2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting
- Author
-
Evelo Chris T, Coort Susan LM, Heim Carolin, Polley Abigael C, Mayer Claus, Horgan Graham, Duthie Susan J, Crosley L Katie, Rubio-Aliaga Isabel, de Roos Baukje, Bouwman Freek G, Mulholland Francis, Johnson Ian T, Elliott Ruan M, Daniel Hannelore, and Mariman Edwin CM
- Subjects
Internal medicine ,RC31-1245 ,Genetics ,QH426-470 - Abstract
Abstract Background Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation. Methods Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach (multiplex immunoassay) were applied to investigate how fasting for 36 h, as compared to 12 h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva collected from ten healthy volunteers. Results Between-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF) analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells. Conclusions Identification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36 h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics.
- Published
- 2011
- Full Text
- View/download PDF
22. The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms
- Author
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Cochrane Brett, Mulholland Francis, Bongaerts Roy JM, Hamilton Shea, Porter Jonathan, Lucchini Sacha, Lappin-Scott Hilary M, and Hinton Jay CD
- Subjects
Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm. Results We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by exogenous tryptophan or indole. Conclusions Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.
- Published
- 2009
- Full Text
- View/download PDF
23. Induction of a chemoattractant transcriptional response by a Campylobacter jejuni boiled cell extract in colonocytes
- Author
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Connerton Phillippa L, Dillon Eleanor, Smith Julie, Clarke Peter, Loughlin Michael F, Connerton Ian F, Mellits Kenneth H, Mulholland Francis, and Hawkey Christopher J
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Microbiology ,QR1-502 - Abstract
Abstract Background Campylobacter jejuni, the commonest cause of bacterial diarrhoea worldwide, can also induce colonic inflammation. To understand how a previously identified heat stable component contributes to pro-inflammatory responses we used microarray and real-time quantitative PCR to investigate the transcriptional response to a boiled cell extract of Campylobacter jejuni NCTC 11168. Results RNA was extracted from the human colonocyte line HCA-7 (clone 29) after incubation for 6 hours with Campylobacter jejuni boiled cell extract and was used to probe the Affymetrix Human Genome U133A array. Genes differentially affected by Campylobacter jejuni boiled cell extract were identified using the Significance Score algorithm of the Bioconductor software suite and further analyzed using the Ingenuity Pathway Analysis program. The chemokines CCL20, CXCL3, CXCL2, Interleukin 8, CXCL1 and CXCL6 comprised 6 of the 10 most highly up-regulated genes, all with Significance Scores ≥ 10. Members of the Tumor Necrosis Factor α/Nuclear Factor-κB super-family were also significantly up-regulated and involved in the most significantly regulated signalling pathways (Death receptor, Interleukin 6, Interleukin 10, Toll like receptor, Peroxisome Proliferator Activated Receptor-γ and apoptosis). Ingenuity Pathway Analysis also identified the most affected functional gene networks such as cell movement, gene expression and cell death. In contrast, down-regulated genes were predominantly concerned with structural and metabolic functions. Conclusion A boiled cell extract of Campylobacter jejuni has components that can directly switch the phenotype of colonic epithelial cells from one of resting metabolism to a pro-inflammatory one, particularly characterized by increased expression of genes for leukocyte chemoattractant molecules.
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- 2009
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24. A PAS domain-containing regulator controls flagella-flagella interactions in Campylobacter jejuni.
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Reuter M, Periago PM, Mulholland F, Brown HL, and van Vliet AH
- Abstract
The bipolar flagella of the foodborne bacterial pathogen Campylobacter jejuni confer motility, which is essential for virulence. The flagella of C. jejuni are post-translationally modified, but how this process is controlled is not well understood. In this work, we have identified a novel PAS-domain containing regulatory system, which modulates flagella-flagella interactions in C. jejuni. Inactivation of the cj1387c gene, encoding a YheO-like PAS6 domain linked to a helix-turn-helix domain, resulted in the generation of a tightly associated "cell-train" morphotype, where up to four cells were connected by their flagella. The morphotype was fully motile, resistant to vortexing, accompanied by increased autoagglutination, and was not observed in aflagellated cells. The Δcj1387c mutant displayed increased expression of the adjacent Cj1388 protein, which comprises of a single endoribonuclease L-PSP domain. Comparative genomics showed that cj1387c (yheO) orthologs in bacterial genomes are commonly linked to an adjacent cj1388 ortholog, with some bacteria, including C. jejuni, containing another cj1388-like gene (cj0327). Inactivation of the cj1388 and cj0327 genes resulted in decreased autoagglutination in Tween-20-supplemented media. The Δcj1388 and Δcj0327 mutants were also attenuated in a Galleria larvae-based infection model. Finally, substituting the sole cysteine in Cj1388 for serine prevented Cj1388 dimerization in non-reducing conditions, and resulted in decreased autoagglutination in the presence of Tween-20. We hypothesize that Cj1388 and Cj0327 modulate post-translational modification of the flagella through yet unidentified mechanisms, and propose naming Cj1387 the Campylobacter Flagella Interaction Regulator CfiR, and the Cj1388 and Cj0327 protein as CfiP and CfiQ, respectively.
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- 2015
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25. PerR controls oxidative stress defence and aerotolerance but not motility-associated phenotypes of Campylobacter jejuni.
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Handley RA, Mulholland F, Reuter M, Ramachandran VK, Musk H, Clissold L, Le Brun NE, and van Vliet AH
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- Bacterial Proteins genetics, Campylobacter jejuni physiology, DNA, Bacterial metabolism, Electrophoretic Mobility Shift Assay, Gene Expression Profiling, Gene Knockout Techniques, Locomotion, Microbial Viability, Mutagenesis, Insertional, Protein Binding, Proteome analysis, Regulon, Repressor Proteins genetics, Transcription Initiation Site, Transcription, Genetic, Bacterial Proteins metabolism, Campylobacter jejuni genetics, Gene Expression Regulation, Bacterial, Oxidative Stress, Repressor Proteins metabolism, Stress, Physiological
- Abstract
The foodborne bacterial pathogen Campylobacter jejuni is an obligate microaerophile that is exposed to atmospheric oxygen during transmission through the food chain. Survival under aerobic conditions requires the concerted control of oxidative stress systems, which in C. jejuni are intimately connected with iron metabolism via the PerR and Fur regulatory proteins. Here, we have characterized the roles of C. jejuni PerR in oxidative stress and motility phenotypes, and its regulon at the level of transcription, protein expression and promoter interactions. Insertional inactivation of perR in the C. jejuni reference strains NCTC 11168, 81-176 and 81116 did not result in any growth deficiencies, but strongly increased survival in atmospheric oxygen conditions, and allowed growth around filter discs infused with up to 30 % H2O2 (8.8 M). Expression of catalase, alkyl hydroperoxide reductase, thioredoxin reductase and the Rrc desulforubrerythrin was increased in the perR mutant, and this was mediated at the transcriptional level as shown by electrophoretic mobility shift assays of the katA, ahpC and trxB promoters using purified PerR. Differential RNA-sequencing analysis of a fur perR mutant allowed the identification of eight previously unknown transcription start sites of genes controlled by Fur and/or PerR. Finally, inactivation of perR in C. jejuni did not result in reduced motility, and did not reduce killing of Galleria melonella wax moth larvae. In conclusion, PerR plays an important role in controlling oxidative stress resistance and aerobic survival of C. jejuni, but this role does not extend into control of motility and associated phenotypes.
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- 2015
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26. Selenium Biomarkers in Prostate Cancer Cell Lines and Influence of Selenium on Invasive Potential of PC3 Cells.
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Hendrickx W, Decock J, Mulholland F, Bao Y, and Fairweather-Tait S
- Abstract
Dietary selenium intake has been linked to reduced cancer risk, however the underlying mechanisms are yet unknown. We question the commonly used practice of applying selenium concentrations found in human blood to in vitro studies and evaluated the utility of biomarkers, e.g., glutathione peroxidase 1 (GPx1) and thioredoxin reductase 1 (TrxR1), to determine appropriate selenium levels for in vitro work. Furthermore, we investigated the effects of Se-methylselenocysteine (SeMSC) on prostate cancer cell migration and invasion. After excluding cytotoxicity, we demonstrated that prostate cancer cell lines respond differently to selenium treatment as observed through biomarker assessment. We found that the maximum levels of GPx1 activity and TrxR1 expression were reached at lower selenium concentrations in LNCaP compared to PC3 cells, and PC3 compared to DU145 cells. Therefore the use of selenium concentrations extrapolated from human studies for in vitro work may be applicable when further informed using a readout of selenium repletion including use of selenium responsive biomarkers. No effect on PC3 migration or invasion was observed after long term SeMSC treatment; however a slight increase was found when treatment was solely administered during the assay. The opposite could be observed when cells were cultured under low serum conditions, with a significant increase in migration upon long term but not upon acute SeMSC treatment. To conclude, these findings indicate that it is imperative to study the selenium sensitivity of an in vitro model preferably using biomarkers before investigating any effects on biological processes, or before comparing models.
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- 2013
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27. Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
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Horn N, Wegmann U, Dertli E, Mulholland F, Collins SR, Waldron KW, Bongaerts RJ, Mayer MJ, and Narbad A
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- Amino Acid Sequence, Bacterial Adhesion genetics, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Carbohydrates analysis, Colony Count, Microbial, Gene Deletion, Genes, Bacterial genetics, Genetic Complementation Test, HT29 Cells, Humans, Lactobacillus growth & development, Lactobacillus ultrastructure, Molecular Sequence Data, Molecular Weight, Multigene Family, Phenotype, Polysaccharides, Bacterial biosynthesis, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Cell Membrane metabolism, Lactobacillus cytology, Lactobacillus genetics, Mutation genetics, Polysaccharides, Bacterial genetics
- Abstract
As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.
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- 2013
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28. Molecular characterization of host-specific biofilm formation in a vertebrate gut symbiont.
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Frese SA, Mackenzie DA, Peterson DA, Schmaltz R, Fangman T, Zhou Y, Zhang C, Benson AK, Cody LA, Mulholland F, Juge N, and Walter J
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- Adhesins, Bacterial metabolism, Animals, Gastrointestinal Tract microbiology, Gene Expression Regulation, Bacterial, Genomics, Limosilactobacillus reuteri growth & development, Mice, Sequence Analysis, DNA, Vertebrates genetics, Vertebrates microbiology, Biofilms growth & development, Host Specificity genetics, Limosilactobacillus reuteri genetics, Symbiosis genetics
- Abstract
Although vertebrates harbor bacterial communities in their gastrointestinal tract whose composition is host-specific, little is known about the mechanisms by which bacterial lineages become selected. The goal of this study was to characterize the ecological processes that mediate host-specificity of the vertebrate gut symbiont Lactobacillus reuteri, and to systematically identify the bacterial factors that are involved. Experiments with monoassociated mice revealed that the ability of L. reuteri to form epithelial biofilms in the mouse forestomach is strictly dependent on the strain's host origin. To unravel the molecular basis for this host-specific biofilm formation, we applied a combination of transcriptome analysis and comparative genomics and identified eleven genes of L. reuteri 100-23 that were predicted to play a role. We then determined expression and importance of these genes during in vivo biofilm formation in monoassociated mice. This analysis revealed that six of the genes were upregulated in vivo, and that genes encoding for proteins involved in epithelial adherence, specialized protein transport, cell aggregation, environmental sensing, and cell lysis contributed to biofilm formation. Inactivation of a serine-rich surface adhesin with a devoted transport system (the SecA2-SecY2 pathway) completely abrogated biofilm formation, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity. In summary, this study established that the epithelial selection of bacterial symbionts in the vertebrate gut can be both specific and highly efficient, resulting in biofilms that are exclusively formed by the coevolved strains, and it allowed insight into the bacterial effectors of this process., Competing Interests: The authors have declared that no competing interests exist.
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- 2013
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29. Selenium-dependent biogenesis of formate dehydrogenase in Campylobacter jejuni is controlled by the fdhTU accessory genes.
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Shaw FL, Mulholland F, Le Gall G, Porcelli I, Hart DJ, Pearson BM, and van Vliet AH
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- Formates metabolism, Gene Deletion, Gene Expression Profiling, Genetic Complementation Test, Magnetic Resonance Spectroscopy, Mutagenesis, Insertional, Promoter Regions, Genetic, Transcription, Genetic, Campylobacter jejuni enzymology, Campylobacter jejuni metabolism, Formate Dehydrogenases metabolism, Gene Expression Regulation, Bacterial, Selenium metabolism
- Abstract
The food-borne bacterial pathogen Campylobacter jejuni efficiently utilizes organic acids such as lactate and formate for energy production. Formate is rapidly metabolized via the activity of the multisubunit formate dehydrogenase (FDH) enzyme, of which the FdhA subunit is predicted to contain a selenocysteine (SeC) amino acid. In this study we investigated the function of the cj1500 and cj1501 genes of C. jejuni, demonstrate that they are involved in selenium-controlled production of FDH, and propose the names fdhT and fdhU, respectively. Insertional inactivation of fdhT or fdhU in C. jejuni resulted in the absence of FdhA and FdhB protein expression, reduced fdhABC RNA levels, the absence of FDH enzyme activity, and the lack of formate utilization, as assessed by (1)H nuclear magnetic resonance. The fdhABC genes are transcribed from a single promoter located two genes upstream of fdhA, and the decrease in fdhABC RNA levels in the fdhU mutant is mediated at the posttranscriptional level. FDH activity and the ability to utilize formate were restored by genetic complementation with fdhU and by supplementation of the growth media with selenium dioxide. Disruption of SeC synthesis by inactivation of the selA and selB genes also resulted in the absence of FDH activity, which could not be restored by selenium supplementation. Comparative genomic analysis suggests a link between the presence of selA and fdhTU orthologs and the predicted presence of SeC in FdhA. The fdhTU genes encode accessory proteins required for FDH expression and activity in C. jejuni, possibly by contributing to acquisition or utilization of selenium.
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- 2012
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30. The response of diatom central carbon metabolism to nitrogen starvation is different from that of green algae and higher plants.
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Hockin NL, Mock T, Mulholland F, Kopriva S, and Malin G
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- Amino Acids metabolism, Aquatic Organisms, Arabidopsis metabolism, Chlamydomonas reinhardtii metabolism, Diatoms physiology, Electrophoresis, Gel, Two-Dimensional, Photosynthesis, Proteins genetics, Proteins metabolism, Carbon metabolism, Diatoms metabolism, Nitrogen metabolism, Proteome
- Abstract
The availability of nitrogen varies greatly in the ocean and limits primary productivity over large areas. Diatoms, a group of phytoplankton that are responsible for about 20% of global carbon fixation, respond rapidly to influxes of nitrate and are highly successful in upwelling regions. Although recent diatom genome projects have highlighted clues to the success of this group, very little is known about their adaptive response to changing environmental conditions. Here, we compare the proteome of the marine diatom Thalassiosira pseudonana (CCMP 1335) at the onset of nitrogen starvation with that of nitrogen-replete cells using two-dimensional gel electrophoresis. In total, 3,310 protein spots were distinguishable, and we identified 42 proteins increasing and 23 decreasing in abundance (greater than 1.5-fold change; P < 0.005). Proteins involved in the metabolism of nitrogen, amino acids, proteins, and carbohydrates, photosynthesis, and chlorophyll biosynthesis were represented. Comparison of our proteomics data with the transcriptome response of this species under similar growth conditions showed good correlation and provided insight into different levels of response. The T. pseudonana response to nitrogen starvation was also compared with that of the higher plant Arabidopsis (Arabidopsis thaliana), the green alga Chlamydomonas reinhardtii, and the cyanobacterium Prochlorococcus marinus. We have found that the response of diatom carbon metabolism to nitrogen starvation is different from that of other photosynthetic eukaryotes and bears closer resemblance to the response of cyanobacteria.
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- 2012
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31. 2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting.
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Bouwman FG, de Roos B, Rubio-Aliaga I, Crosley LK, Duthie SJ, Mayer C, Horgan G, Polley AC, Heim C, Coort SL, Evelo CT, Mulholland F, Johnson IT, Elliott RM, Daniel H, and Mariman EC
- Subjects
- Apolipoproteins A blood, Biomarkers metabolism, Cluster Analysis, Electrophoresis, Gel, Two-Dimensional, Guanine Nucleotide Dissociation Inhibitors metabolism, Humans, Immunoassay, Leptin metabolism, Mass Spectrometry, Matrix Metalloproteinase 3 metabolism, Principal Component Analysis, Time Factors, Tumor Suppressor Proteins metabolism, rho Guanine Nucleotide Dissociation Inhibitor beta, rho-Specific Guanine Nucleotide Dissociation Inhibitors, Blood Cells metabolism, Body Fluids metabolism, Fasting, Proteome metabolism, Proteomics methods
- Abstract
Background: Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation., Methods: Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach (multiplex immunoassay) were applied to investigate how fasting for 36 h, as compared to 12 h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva collected from ten healthy volunteers., Results: Between-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF) analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells., Conclusions: Identification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36 h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics., (© 2011 Bouwman et al; licensee BioMed Central Ltd.)
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- 2011
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32. Roles of the twin-arginine translocase and associated chaperones in the biogenesis of the electron transport chains of the human pathogen Campylobacter jejuni.
- Author
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Hitchcock A, Hall SJ, Myers JD, Mulholland F, Jones MA, and Kelly DJ
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- Animals, Bacterial Proteins genetics, Campylobacter jejuni genetics, Chickens, DNA, Bacterial genetics, Electron Transport, Electron Transport Chain Complex Proteins metabolism, Genetic Complementation Test, Membrane Transport Proteins genetics, Molecular Chaperones genetics, Mutation, Nitric Oxide metabolism, Nitrites metabolism, Proteome metabolism, Bacterial Proteins metabolism, Campylobacter jejuni enzymology, Membrane Transport Proteins metabolism, Molecular Chaperones metabolism
- Abstract
The zoonotic pathogen Campylobacter jejuni NCTC 11168 uses a complex set of electron transport chains to ensure growth with a variety of electron donors and alternative electron acceptors, some of which are known to be important for host colonization. Many of the key redox proteins essential for electron transfer in this bacterium have N-terminal twin-arginine translocase (TAT) signal sequences that ensure their transport across the cytoplasmic membrane in a folded state. By comparisons of 2D gels of periplasmic extracts, gene fusions and specific enzyme assays in wild-type, tatC mutant and complemented strains, we experimentally verified the TAT dependence of 10 proteins with an N-terminal twin-arginine motif. NrfH, which has a TAT-like motif (LRRKILK), was functional in nitrite reduction in a tatC mutant, and was correctly rejected as a TAT substrate by the tatfind and TatP prediction programs. However, the hydrogenase subunit HydA is also rejected by tatfind, but was shown to be TAT-dependent experimentally. The YedY homologue Cj0379 is the only TAT translocated molybdoenzyme of unknown function in C. jejuni; we show that a cj0379c mutant is deficient in chicken colonization and has a nitrosative stress phenotype, suggestive of a possible role for Cj0379 in the reduction of reactive nitrogen species in the periplasm. Only two potential TAT chaperones, NapD and Cj1514, are encoded in the genome. Surprisingly, despite homology to TorD, Cj1514 was shown to be specifically required for the activity of formate dehydrogenase, not trimethylamine N-oxide reductase, and was designated FdhM.
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- 2010
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33. The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms.
- Author
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Hamilton S, Bongaerts RJ, Mulholland F, Cochrane B, Porter J, Lucchini S, Lappin-Scott HM, and Hinton JC
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- Bacterial Adhesion, Gene Deletion, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genomic Islands, HeLa Cells, Humans, Oligonucleotide Array Sequence Analysis, Proteome genetics, RNA, Bacterial genetics, Salmonella typhimurium growth & development, Salmonella typhimurium metabolism, Biofilms growth & development, Gene Expression Profiling, Salmonella typhimurium genetics, Tryptophan metabolism
- Abstract
Background: Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm., Results: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by exogenous tryptophan or indole., Conclusions: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.
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- 2009
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34. Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics.
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Crosley LK, Duthie SJ, Polley AC, Bouwman FG, Heim C, Mulholland F, Horgan G, Johnson IT, Mariman EC, Elliott RM, Daniel H, and de Roos B
- Abstract
Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400-1,500 mug protein from platelets, and 100-600 mug from PBMC. 30 muL plasma depleted of albumin and IgG provided 350-650 mug protein. A sample of morning urine provided 0.9-8.6 mg protein/dL, and a sample of saliva provided 70-950 mug protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.
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- 2009
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35. Induction of a chemoattractant transcriptional response by a Campylobacter jejuni boiled cell extract in colonocytes.
- Author
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Mellits KH, Connerton IF, Loughlin MF, Clarke P, Smith J, Dillon E, Connerton PL, Mulholland F, and Hawkey CJ
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- Cell Line, Chemokines biosynthesis, Chemotactic Factors isolation & purification, Colon cytology, Down-Regulation, Humans, Oligonucleotide Array Sequence Analysis, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction immunology, Up-Regulation, Campylobacter jejuni chemistry, Campylobacter jejuni immunology, Chemotactic Factors immunology, Colon immunology, Epithelial Cells immunology, Gene Expression Profiling
- Abstract
Background: Campylobacter jejuni, the commonest cause of bacterial diarrhoea worldwide, can also induce colonic inflammation. To understand how a previously identified heat stable component contributes to pro-inflammatory responses we used microarray and real-time quantitative PCR to investigate the transcriptional response to a boiled cell extract of Campylobacter jejuni NCTC 11168., Results: RNA was extracted from the human colonocyte line HCA-7 (clone 29) after incubation for 6 hours with Campylobacter jejuni boiled cell extract and was used to probe the Affymetrix Human Genome U133A array. Genes differentially affected by Campylobacter jejuni boiled cell extract were identified using the Significance Score algorithm of the Bioconductor software suite and further analyzed using the Ingenuity Pathway Analysis program. The chemokines CCL20, CXCL3, CXCL2, Interleukin 8, CXCL1 and CXCL6 comprised 6 of the 10 most highly up-regulated genes, all with Significance Scores > or = 10. Members of the Tumor Necrosis Factor alpha/Nuclear Factor-kappaB super-family were also significantly up-regulated and involved in the most significantly regulated signalling pathways (Death receptor, Interleukin 6, Interleukin 10, Toll like receptor, Peroxisome Proliferator Activated Receptor-gamma and apoptosis). Ingenuity Pathway Analysis also identified the most affected functional gene networks such as cell movement, gene expression and cell death. In contrast, down-regulated genes were predominantly concerned with structural and metabolic functions., Conclusion: A boiled cell extract of Campylobacter jejuni has components that can directly switch the phenotype of colonic epithelial cells from one of resting metabolism to a pro-inflammatory one, particularly characterized by increased expression of genes for leukocyte chemoattractant molecules.
- Published
- 2009
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36. Oxygen- and NssR-dependent globin expression and enhanced iron acquisition in the response of campylobacter to nitrosative stress.
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Monk CE, Pearson BM, Mulholland F, Smith HK, and Poole RK
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- Electrophoresis, Gel, Two-Dimensional, Gene Expression Regulation, Bacterial, Iron chemistry, Iron metabolism, Models, Biological, Mutation, Nitric Oxide chemistry, Nitrogen chemistry, Oxygen chemistry, Proteomics methods, Time Factors, Transcription Factors genetics, Transcription, Genetic, Bacterial Proteins genetics, Bacterial Proteins physiology, Campylobacter jejuni metabolism, Gene Expression Regulation, Globins metabolism, Nitrogen metabolism, Oxygen metabolism, Transcription Factors physiology, Truncated Hemoglobins genetics, Truncated Hemoglobins physiology
- Abstract
Pathogenic bacteria experience nitrosative stress from NO generated in the host and from nitrosating species such as S-nitrosoglutathione. The food-borne pathogen Campylobacter jejuni responds by activating gene expression from a small regulon under the control of the NO-sensitive regulator, NssR. Here, we describe the full extent of the S-nitrosoglutathione response using transcriptomic and proteomic analysis of batch- and chemostat-cultured C. jejuni. In addition to the NssR regulon, which includes two hemoglobins (Cgb and Ctb), we identify more than 90 other up-regulated genes, notably those encoding heat shock proteins and proteins involved in oxidative stress tolerance and iron metabolism/transport. Up-regulation of a subset of these genes, including cgb, is also elicited by NO-releasing compounds. Mutation of the iron-responsive regulator Fur results in insensitivity of growth to NO, suggesting that derepression of iron-regulated genes and augmentation of iron acquisition is a physiological response to nitrosative damage. We describe the effect of oxygen availability on nitrosative stress tolerance; cells cultured at higher rates of oxygen diffusion have elevated levels of hemoglobins, are more resistant to inhibition by NO of both growth and respiration, and consume NO more rapidly. The oxygen response is mediated by NssR. Thus, in addition to NO detoxification catalyzed by the hemoglobins Cgb and possibly Ctb, C. jejuni mounts an extensive stress response. We suggest that inhibition of respiration by NO may increase availability of oxygen for Cgb synthesis and function.
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- 2008
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37. Serotonin receptors, novel targets of sulforaphane identified by proteomic analysis in Caco-2 cells.
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Mastrangelo L, Cassidy A, Mulholland F, Wang W, and Bao Y
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- Adenocarcinoma drug therapy, Adenocarcinoma metabolism, Biomarkers, Pharmacological analysis, Biomarkers, Tumor analysis, Biomarkers, Tumor isolation & purification, Biomarkers, Tumor metabolism, Caco-2 Cells, Colonic Neoplasms drug therapy, Colonic Neoplasms metabolism, Drug Delivery Systems, Drug Screening Assays, Antitumor, Humans, Isothiocyanates, Proteome analysis, Receptors, Serotonin analysis, Receptors, Serotonin metabolism, Serotonin Antagonists pharmacology, Sulfoxides, Thiocyanates therapeutic use, Proteomics, Receptors, Serotonin isolation & purification, Serotonin Antagonists isolation & purification, Thiocyanates pharmacology
- Abstract
Cancer chemopreventive activity of sulforaphane has been predominantly associated with its ability to induce phase II detoxification enzymes. In the present study, novel targets of sulforaphane were identified and characterized using a proteomics approach. Two-dimensional gel electrophoresis and mass spectrometry were used to produce protein profiles of human colon adenocarcinoma Caco-2 cells treated with 5 mumol/L sulforaphane for 48 h and control cells (0.05% DMSO). Gel comparisons showed the down-regulation to undetectable level of the serotonin receptor 5-HT(3) after sulforaphane treatment. In addition, Aldo-keto reductase and d-dopachrome decarboxylase were also differentially expressed in control and treated cell extracts. To elucidate two-dimensional gel findings, the neurotransmitter receptors 5-HT(3A), 5-HT(1A), 5-HT(2C), and the serotonin reuptake transporter were analyzed using Western blotting. Results showed a decrease of neurotransmitter receptors in a dose-dependent manner after sulforaphane treatment. Moreover, after exposure of Caco-2 cells to sulforaphane, nicotinic acetylcholine receptor protein level was increased. These findings suggested a potential effect of sulforaphane on serotonin release. Activation of neurotransmitter receptors followed by initiation of cyclic AMP signaling might be crucial events in colon carcinoma progression. Thus, the effect of sulforaphane may help to elucidate signaling pathways serotonin-mediated in colon cancer and lead to development of potential novel therapeutic agents.
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- 2008
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38. Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate.
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Guccione E, Leon-Kempis Mdel R, Pearson BM, Hitchin E, Mulholland F, van Diemen PM, Stevens MP, and Kelly DJ
- Subjects
- Aerobiosis, Animals, Aspartate Aminotransferases genetics, Aspartate Ammonia-Lyase chemistry, Aspartate Ammonia-Lyase genetics, Aspartic Acid metabolism, Bacterial Proteins genetics, Biosynthetic Pathways, Campylobacter Infections microbiology, Campylobacter Infections veterinary, Campylobacter jejuni chemistry, Campylobacter jejuni genetics, Chickens, Culture Media chemistry, Dicarboxylic Acid Transporters genetics, Dicarboxylic Acid Transporters metabolism, Fumarates metabolism, Glutamic Acid metabolism, Humans, Kinetics, Mutation, Transcription, Genetic, Amino Acids metabolism, Aspartate Aminotransferases metabolism, Aspartate Ammonia-Lyase metabolism, Bacterial Proteins metabolism, Campylobacter jejuni enzymology, Campylobacter jejuni growth & development, Oxygen metabolism
- Abstract
Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni. During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilize any amino acid except serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA-dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.
- Published
- 2008
- Full Text
- View/download PDF
39. Riboflavin biosynthesis is associated with assimilatory ferric reduction and iron acquisition by Campylobacter jejuni.
- Author
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Crossley RA, Gaskin DJ, Holmes K, Mulholland F, Wells JM, Kelly DJ, van Vliet AH, and Walton NJ
- Subjects
- Bacterial Proteins metabolism, Diacetyl metabolism, Flavin Mononucleotide metabolism, Flavin-Adenine Dinucleotide metabolism, Gene Deletion, Intramolecular Transferases genetics, Oxidation-Reduction, Repressor Proteins metabolism, Campylobacter jejuni metabolism, Ferric Compounds metabolism, Iron metabolism, Riboflavin biosynthesis
- Abstract
One of the pathways involved in the acquisition of the essential metal iron by bacteria involves the reduction of insoluble Fe(3+) to soluble Fe(2+), followed by transport of Fe(2+) to the cytoplasm. Flavins have been implicated as electron donors in this poorly understood process. Ferrous iron uptake is essential for intestinal colonization by the important pathogen Campylobacter jejuni and may be of particular importance under low-oxygen conditions. In this study, the links among riboflavin biosynthesis, ferric reduction, and iron acquisition in C. jejuni NCTC11168 have been investigated. A riboflavin auxotroph was generated by inactivation of the ribB riboflavin biosynthesis gene (Cj0572), and the resulting isogenic ribB mutant only grew in the presence of exogenous riboflavin or the riboflavin precursor diacetyl but not in the presence of the downstream products flavin adenine dinucleotide and flavin mononucleotide. Riboflavin uptake was unaffected in the ribB mutant under iron-limited conditions but was lower in both the wild-type strain and the ribB mutant under iron-replete conditions. Mutation of the fur gene, which encodes an iron uptake regulator of C. jejuni, resulted in an increase in riboflavin uptake which was independent of the iron content of the medium, suggesting a role for Fur in the regulation of the as-yet-unknown riboflavin transport system. Finally, ferric reduction activity was independent of iron availability in the growth medium but was lowered in the ribB mutant compared to the wild-type strain and, conversely, increased in the fur mutant. Taken together, the findings confirm close relationships among iron acquisition, riboflavin production, and riboflavin uptake in C. jejuni.
- Published
- 2007
- Full Text
- View/download PDF
40. Contribution of conserved ATP-dependent proteases of Campylobacter jejuni to stress tolerance and virulence.
- Author
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Cohn MT, Ingmer H, Mulholland F, Jørgensen K, Wells JM, and Brøndsted L
- Subjects
- ATP-Dependent Proteases genetics, Adaptation, Physiological, Bacterial Adhesion genetics, Bacterial Adhesion physiology, Bacterial Proteins metabolism, Cell Line, Endopeptidase Clp genetics, Endopeptidase Clp physiology, Epithelial Cells microbiology, Gene Deletion, Hot Temperature, Humans, Locomotion genetics, Locomotion physiology, Peptide Hydrolases genetics, Peptide Hydrolases physiology, Protease La genetics, Protease La physiology, Protein Folding, Virulence, ATP-Dependent Proteases physiology, Campylobacter jejuni pathogenicity, Campylobacter jejuni physiology
- Abstract
In prokaryotic cells the ATP-dependent proteases Lon and ClpP (Clp proteolytic subunit) are involved in the turnover of misfolded proteins and the degradation of regulatory proteins, and depending on the organism, these proteases contribute variably to stress tolerance. We constructed mutants in the lon and clpP genes of the food-borne human pathogen Campylobacter jejuni and found that the growth of both mutants was impaired at high temperature, a condition known to increase the level of misfolded protein. Moreover, the amounts of misfolded protein aggregates were increased when both proteases were absent, and we propose that both ClpP and Lon are involved in eliminating misfolded proteins in C. jejuni. In order to bind misfolded protein, ClpP has to associate with one of several Clp ATPases. Following inactivation of the ATPase genes clpA and clpX, only the clpX mutant displayed the same heat sensitivity as the clpP mutant, indicating that the ClpXP proteolytic complex is responsible for the degradation of heat-damaged proteins in C. jejuni. Notably, ClpP and ClpX are required for growth at 42 degrees C, which is the temperature of the intestinal tract of poultry, one of the primary carriers of C. jejuni. Thus, ClpP and ClpX may be suitable targets of new intervention strategies aimed at reducing C. jejuni in poultry production. Further characterization of the clpP and lon mutants revealed other altered phenotypes, such as reduced motility, less autoagglutination, and lower levels of invasion of INT407 epithelial cells, suggesting that the proteases may contribute to the virulence of C. jejuni.
- Published
- 2007
- Full Text
- View/download PDF
41. Proteomic analysis reveals field-wide changes in protein expression in the morphologically normal mucosa of patients with colorectal neoplasia.
- Author
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Polley AC, Mulholland F, Pin C, Williams EA, Bradburn DM, Mills SJ, Mathers JC, and Johnson IT
- Subjects
- Aged, Aged, 80 and over, Amino Acid Sequence, Colorectal Neoplasms chemistry, Colorectal Neoplasms pathology, Electrophoresis, Gel, Two-Dimensional, Female, Humans, Intestinal Mucosa chemistry, Male, Middle Aged, Molecular Sequence Data, Neoplasm Proteins analysis, Neoplasm Staging, Proteomics methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Colorectal Neoplasms metabolism, Intestinal Mucosa metabolism, Neoplasm Proteins biosynthesis
- Abstract
Models for the pathogenesis of colorectal cancer tend to focus on the localized lesion, with less attention paid to changes in normal-appearing mucosa. Here we used two-dimensional gel electrophoresis and mass spectrometry to define patterns of protein expression in morphologically normal colonic mucosa from 13 healthy subjects, 9 patients with adenomatous polyps, and 9 with cancer. Tumor samples were also compared with the normal mucosa. Systematic gel comparisons identified a total of 839 spots that differed significantly between one or more groups (P < 0.05). Principle component analysis indicated that the first three components accounted for approximately 37% of the total variation and provided clear evidence that flat mucosa from healthy subjects differed significantly from that of patients with polyps or cancer. Sixty-one proteins differed significantly between mucosa from healthy subjects and all other tissue types, and 206 differed significantly between healthy mucosa and polyp mucosa. Several of the proteins showing significant underexpression in tumor tissue were cytokeratins and other cytoskeletal components. In contrast, cytokeratins, including several isoforms of cytokeratin 8, were overexpressed in apparently normal mucosa from polyp and cancer patients compared with mucosa from healthy subjects. These findings indicate that protein expression in the apparently normal colonic mucosal field is modified in individuals with neoplastic lesions at sites distant from the lesion. Recognition and further characterization of this field effect at the molecular level may provide protein biomarkers of susceptibility to colorectal cancer and facilitate development of hypotheses for the role of diet and other environmental factors in its causation.
- Published
- 2006
- Full Text
- View/download PDF
42. The Campylobacter jejuni PEB1a adhesin is an aspartate/glutamate-binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids.
- Author
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Leon-Kempis Mdel R, Guccione E, Mulholland F, Williamson MP, and Kelly DJ
- Subjects
- ATP-Binding Cassette Transporters genetics, Animals, Antigens, Bacterial genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Campylobacter jejuni cytology, Campylobacter jejuni genetics, Culture Media chemistry, Humans, Recombinant Proteins genetics, Recombinant Proteins metabolism, Subcellular Fractions chemistry, ATP-Binding Cassette Transporters metabolism, Aerobiosis physiology, Amino Acids, Dicarboxylic metabolism, Antigens, Bacterial metabolism, Aspartic Acid metabolism, Campylobacter jejuni physiology, Glutamic Acid metabolism
- Abstract
The PEB1a protein of the gastrointestinal pathogen Campylobacter jejuni mediates interactions with epithelial cells and is an important factor in host colonization. Cell fractionation and immunoblotting showed that PEB1a is most abundant in the periplasm of C. jejuni, and is detectable in the culture supernatant but not in the inner or outer membrane. The protein is homologous with periplasmic-binding proteins associated with ABC transporters and we show by fluorescence spectroscopy that purified recombinant PEB1a binds L-aspartate and L-glutamate with sub microM K(d) values. Binding of L-14C-aspartate or L-14C-glutamate was strongly out-competed by excess unlabelled aspartate or glutamate but only poorly by asparagine and glutamine. A mutant in the Cj0921c gene, encoding PEB1a, was completely unable to transport 5 microM L-14C-glutamate and showed a large reduction (approximately 20-fold) in the rate of L-14C-aspartate transport compared with the wild type. Although microaerobic growth of this mutant was little affected in complex media, growth on aspartate or glutamate in defined media was completely prevented, whereas growth with serine was similar to wild type. 1H-NMR analysis of the culture supernatants of the Cj0921c mutant showed some utilization of aspartate but not glutamate, consistent with the transport data. It is concluded that in addition to the established role of PEB1a as an adhesin, the PEB1 transport system plays a key role in the utilization of aspartate and glutamate, which may be important in vivo carbon sources for this pathogen.
- Published
- 2006
- Full Text
- View/download PDF
43. Evidence that the essential response regulator YycF in Streptococcus pneumoniae modulates expression of fatty acid biosynthesis genes and alters membrane composition.
- Author
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Mohedano ML, Overweg K, de la Fuente A, Reuter M, Altabe S, Mulholland F, de Mendoza D, López P, and Wells JM
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial, Microarray Analysis, Proteomics, Signal Transduction, Streptococcus pneumoniae genetics, Transcription, Genetic, Bacterial Proteins physiology, Cell Membrane metabolism, Fatty Acids biosynthesis, Streptococcus pneumoniae metabolism
- Abstract
The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism.
- Published
- 2005
- Full Text
- View/download PDF
44. Diverse roles for HspR in Campylobacter jejuni revealed by the proteome, transcriptome and phenotypic characterization of an hspR mutant.
- Author
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Andersen MT, Brøndsted L, Pearson BM, Mulholland F, Parker M, Pin C, Wells JM, and Ingmer H
- Subjects
- Bacterial Proteins genetics, Campylobacter jejuni genetics, Campylobacter jejuni metabolism, Campylobacter jejuni pathogenicity, Heat-Shock Proteins genetics, Heat-Shock Response, Humans, Movement, Mutation, Phenotype, Proteome, Repressor Proteins genetics, Virulence, Bacterial Proteins metabolism, Campylobacter jejuni physiology, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Heat-Shock Proteins metabolism, Oligonucleotide Array Sequence Analysis methods, Repressor Proteins metabolism, Transcription, Genetic
- Abstract
Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. The role of a homologue of the negative transcriptional regulatory protein HspR, which in other organisms participates in the control of the heat-shock response, was investigated. Following inactivation of hspR in C. jejuni, members of the HspR regulon were identified by DNA microarray transcript profiling. In agreement with the predicted role of HspR as a negative regulator of genes involved in the heat-shock response, it was observed that the transcript amounts of 13 genes were increased in the hspR mutant, including the chaperone genes dnaK, grpE and clpB, and a gene encoding the heat-shock regulator HrcA. Proteomic analysis also revealed increased synthesis of the heat-shock proteins DnaK, GrpE, GroEL and GroES in the absence of HspR. The altered expression of chaperones was accompanied by heat sensitivity, as the hspR mutant was unable to form colonies at 44 degrees C. Surprisingly, transcriptome analysis also revealed a group of 17 genes with lower transcript levels in the hspR mutant. Of these, eight were predicted to be involved in the formation of the flagella apparatus, and the decreased expression is likely to be responsible for the reduced motility and ability to autoagglutinate that was observed for hspR mutant cells. Electron micrographs showed that mutant cells were spiral-shaped and carried intact flagella, but were elongated compared to wild-type cells. The inactivation of hspR also reduced the ability of Campylobacter to adhere to and invade human epithelial INT-407 cells in vitro, possibly as a consequence of the reduced motility or lower expression of the flagellar export apparatus in hspR mutant cells. It was concluded that, in C. jejuni, HspR influences the expression of several genes that are likely to have an impact on the ability of the bacterium to successfully survive in food products and subsequently infect the consumer.
- Published
- 2005
- Full Text
- View/download PDF
45. Campylobacter jejuni gene expression in response to iron limitation and the role of Fur.
- Author
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Holmes K, Mulholland F, Pearson BM, Pin C, McNicholl-Kennedy J, Ketley JM, and Wells JM
- Subjects
- Animals, Bacterial Proteins genetics, Campylobacter jejuni genetics, Gene Expression Profiling, Humans, Mutation, Oligonucleotide Array Sequence Analysis, Promoter Regions, Genetic, Proteome, Transcription, Genetic, Bacterial Proteins metabolism, Campylobacter jejuni growth & development, Campylobacter jejuni metabolism, Gene Expression Regulation, Bacterial, Iron metabolism, Repressor Proteins metabolism
- Abstract
Campylobacter jejuni is a zoonotic pathogen and the most common cause of bacterial foodborne diarrhoeal illness worldwide. To establish intestinal colonization prior to either a commensal or pathogenic interaction with the host, C. jejuni will encounter iron-limited niches where there is likely to be intense competition from the host and normal microbiota for iron. To gain a better understanding of iron homeostasis and the role of ferric uptake regulator (Fur) in iron acquisition in C. jejuni, a proteomic and transcriptome analysis of wild-type and fur mutant strains in iron-rich and iron-limited growth conditions was carried out. All of the proposed iron-transport systems for haemin, ferric iron and enterochelin, as well as the putative iron-transport genes p19, Cj1658, Cj0177, Cj0178 and cfrA, were expressed at higher levels in the wild-type strain under iron limitation and in the fur mutant in iron-rich conditions, suggesting that they were regulated by Fur. Genes encoding a previously uncharacterized ABC transport system (Cj1660-Cj1663) also appeared to be Fur regulated, supporting a role for these genes in iron uptake. Several promoters containing consensus Fur boxes that were identified in a previous bioinformatics search appeared not to be regulated by iron or Fur, indicating that the Fur box consensus needs experimental refinement. Binding of purified Fur to the promoters upstream of the p19, CfrA and CeuB operons was verified using an electrophoretic mobility shift assay (EMSA). These results also implicated Fur as having a role in the regulation of several genes, including fumarate hydratase, that showed decreased expression in response to iron limitation. The known PerR promoters were also derepressed in the C. jejuni Fur mutant, suggesting that they might be co-regulated in response to iron and peroxide stress. These results provide new insights into the effects of iron on metabolism and oxidative stress response as well as the regulatory role of Fur.
- Published
- 2005
- Full Text
- View/download PDF
46. The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide.
- Author
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Almirón-Roig E, Mulholland F, Gasson MJ, and Griffin AM
- Subjects
- Base Sequence, Chromosomes, Bacterial genetics, Cloning, Molecular, DNA Primers genetics, DNA Transposable Elements genetics, Escherichia coli genetics, Gene Expression, Glycosyltransferases genetics, Molecular Sequence Data, Open Reading Frames, Genes, Bacterial, Multigene Family, Polysaccharides, Bacterial biosynthesis, Streptococcus genetics, Streptococcus metabolism
- Abstract
The cpsFGHIJKL genes from the cps cluster of Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of EPS were identified, cloned and nucleotide sequenced. The complete cps cluster is contained on an approximately 11.2 kb chromosomal region which contains 12 ORFs, including the previously cloned cpsABCDE genes. Functions were assigned to some of the predicted gene products on the basis of homology to known sequences as follows: cpsK encodes a protein thought to be involved in the polymerization and export of the polysaccharide; cpsE, cpsF, cpsG, cpsH, cpsI and cpsJ encode putative sugar transferases. Two insertion sequences, IS1193 and ISS1, were identified within and flanking the 3' end of the cps cluster respectively. Analysis of the expression of the cpsE gene in Escherichia coli demonstrated that it encodes a glucose-1-phosphate transferase; the enzyme which catalyses the first step in EPS biosynthesis in S. thermophilus NCFB 2393.
- Published
- 2000
- Full Text
- View/download PDF
47. A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk.
- Author
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l'Anson KJ, Movahedi S, Griffin HG, Gasson MJ, and Mulholland F
- Subjects
- Amino Acid Sequence, Aminopeptidases genetics, Animals, Base Sequence, Cellulase genetics, Cloning, Molecular, Clostridium enzymology, Clostridium genetics, DNA Primers genetics, DNA, Bacterial genetics, Escherichia coli genetics, Gene Library, Glutamyl Aminopeptidase, Lactococcus lactis genetics, Molecular Sequence Data, Mutagenesis, Open Reading Frames, Sequence Homology, Amino Acid, Aminopeptidases metabolism, Lactococcus lactis enzymology, Lactococcus lactis growth & development, Milk microbiology
- Abstract
Degenerate PCR primers were designed from the N-terminal amino acid sequence of a glutamyl aminopeptidase (PepA) from Lactococcus lactis. These primers were used to screen a lambda library for clones containing the gene (pepA) encoding PepA. The DNA sequence of a 2.1 kb fragment containing pepA was determined. The sequence revealed the presence of one complete and two incomplete open reading frames (ORFs). The complete ORF encodes a putative protein of 353 amino acids with a predicted N-terminal sequence identical to that determined for purified PepA. The pepA gene was subcloned on an Escherichia coli plasmid vector and production of active PepA was confirmed by means of a zymogram. Mutants of L. lactis in which the pepA gene was inactivated grew to normal cell densities in milk but exhibited a reduced growth rate during the exponential phase. Thus whilst PepA is required for optimal growth it is not essential.
- Published
- 1995
- Full Text
- View/download PDF
48. The influence of biotechnological developments on cheese manufacture.
- Author
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Law BA and Mulholland F
- Subjects
- Animals, Cattle, Chymosin biosynthesis, Chymosin genetics, Endopeptidases genetics, Endopeptidases metabolism, Food Handling methods, Food Microbiology, Food Preservation methods, Lactococcus lactis growth & development, Protein Engineering, Recombinant Proteins biosynthesis, Biotechnology methods, Cheese
- Published
- 1991
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