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Modulation of Phosphoinositide Metabolism by Intracellular Pathogenic Bacteria Listeria monocytogenes

Modulation of Phosphoinositide Metabolism by Intracellular Pathogenic Bacteria Listeria monocytogenes A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty
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Modulation of Phosphoinositide Metabolism by Intracellular Pathogenic Bacteria Listeria monocytogenes A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2012 Jiahui Wang List of Contents List of Contents... 2 List of Figures... 6 List of Tables Abstract Declaration Copyright Statement Acknowledgement Abbreviations Preface Chapter 1 Introduction Listeria monocytogenes a food borne pathogen Taxonomy Diseases caused by L. monocytogenes Listeriosis in animals Listeriosis in humans Foetomaternal and neonatal listeriosis Listeriosis in adult Pathogenesis of infection and intracellular life cycle of L. monocytogenes Internalisation Internalin A Internalin B Other proteins related to internalisation Escape from phagosome LLO Phospholipase C Cytosolic growth Actin-based motility and cell-cell spread Organisation and regulation of L. monocytogenes virulence genes Infection route of L. monocytogenes in humans Immunity against L. monocytogenes infection and immune-invasion of L. monocytogenes Innate immunity to L. monocytogenes infection Adaptive immunity against L. monocytogenes infection Immune invasion of L. monocytogenes Phosphoinositide metabolism in eukaryotic cells PIPs in plasma membrane dynamics PIPs in endosomal dynamics and intracellular membrane traffic PIPs in the nucleus PI3K signaling pathway Modulation of host cell PIP metabolism by pathogenic bacteria Shigella flexneri S. enterica [2] 1.9.3 Mycobacterium tuberculosis Legionella pneumophila L. monocytogenes LipA and LipB two tyrosine and lipid phosphatases produced by L. monocytogenes Aim of the project Chapter 2 Methods and Materials Bacteria strains, media and growth conditions Plasmids DNA manipulation Preparation of plasmid DNA from E. coli Restriction endonuclease digestion of DNA Purification of restriction digests and PCR products Ligation reactions Agarose gel electrophoresis Gel purification of DNA fragments Polymerase chain reaction (PCR) Colony PCR Quantificatio of DNA DNA sequencing Cloning with the pgem-t Easy vector system Transformation of DNA into bacteria Preparation of chemically competent E.coli Preparation of electrocompetent L. monocytogenes Transformation of plasmid DNA into chemically competent E. coli Transformation of plasmid DNA into electrocompetent L. monocytogenes Site-directed mutagenesis Mutagenesis in L. monocytogenes via induced homologous recombination Mammalian cell culture Mammalian cell lines and culture conditions Freeze down and revival of mammalian cells Transient transfection of plasmid DNA into Hela M cells Intracellular survival assay of L. monocytogenes Immunofluorescent staining and imaging Plaque assay Quantification of fluorescence expression Calculation of mean doubling time of L. monocytogenes Chapter 3 L. monocytogenes EGDe::ΔlipA and EGDe::ΔlipB mutants are attenuated in intracellular growth in Hela M cells in vitro Introduction Determination of the optimal conditions of intracellular survival assay in Hela M cells L. monocytogenes EGDe, EGDe::ΔlipA, EGDe::ΔlipB mutants were attenuated in intracellular growth in Hela M cells Discussion [3] Chapter 4 L. monocytogenes InlA::ΔlipA and InlA::ΔlipB mutants are attenuated in intracellular growth in vitro Introduction InlA strain was unaffected in intracellular growth in Hela M cells Generation of deletion mutations in the InlA strain Deletion of lipa caused attenuation in intracellular growth of L. monocytogenes in Caco-2 cells InlA::ΔlipA was attenuated in intracellular growth in Caco-2 cells Plasmid complementation restored the intracellular growth of InlA::ΔlipA in Caco-2 cells lipa and lipb deletion mutants were attenuated in intracellular growth in Hela M cells InlA::ΔplcBΔhly was defect in intracellular growth Hela M cells lipa and lipb deletion mutants were attenuated in intracellular growth in Hela M cell Plasmid complementation restored the virulence of InlA::ΔlipB in Hela M cells Deletion of lipa and lipb did not affect the intracellular survival of L. monocytogenes in J774.A1 cells lipa and lipb deletion mutants were unaffected in intracellular mobility but were attenuated in forming plaques InlA::ΔlipA and InlA::ΔlipB were unaffected in actin polymerisation and actin-based intracellular movement InlA::ΔlipA, InlA::ΔlipB and InlA::ΔlipAΔlipB formed reduced number of plaques in plaque assay in Hela M cells InlA::ΔlipA, InlA::ΔlipB, InlA::ΔlipAΔlipB were not attenuated in internalisation into Hela M cells at low MOI Discussion The use of the InlA strain Infection of Caco-2, Hela M and J774.A1 cells by L. monocytogenes InlA::ΔlipA, InlA::ΔlipB and InlA::ΔlipAΔlipB were attenuated in intracellular growth LipA and LipB were not required in actin polymerisation Chapter 5 Modulation of host PIP metabolism by L. monocytogenes in Hela M cells Introduction Visualisation of L. monocytogenes Chromosomal expression of fluorescent proteins in L. monocytogenes Generation of fluorescent L. monocytogenes strain InlA::GFP uv and InlA::mCherry InlA::GFP uv has the same growth rate in TSB and in Hela M cell as the InlA strain Expression and visualisation of GFP uv and mcherry in L. monocytogenes Plasmid expression of fluorescent proteins in L. monocytogenes Construction of punk1-prpob-mcherry, punk1-prpob-gfp mut [4] Fluorescence level expressed from punk1-prpob-mcherry in E. coli and L. monocytogenes Expression of fluorescent protein from pnf8 and pjeban6 in L. monocytogenes Antibody staining of L. monocytogenes Localisation of five PIPs in Hela M cells during L. monocytogenes infection Localisation of five PIPs in Hela M cells during L. monocytogenes InlA infection InlA recruited PI3P to its surface in early stage of infection L. monocytogenes did not affect the localisation of PI4P L. monocytogenes did not affect the localisation of PI(3,4,5)P L. monocytogenes InlA co-localised with PI(3,4)P 2 in late stages of infection L. monocytogenes did not affect the localisation of YFP-Akt-PH Localisation of five PIPs in Hela M cells during infection with L. monocytogenes lipa and lipb mutants lipa and lipb mutants did not affect the localisation of PI3P, PI4P, PI(4,5)P 2 and PI(3,4,5)P ΔlipA and ΔlipAΔlipB co-localised with PI(3,4)P 2 at a lower ratio than InlA The localisation of LipA and LipB proteins in Hela M cells Over-expression of LipA and LipB with C-terminal GFP tag Over-expression of inactivated LipA and LipB with C-terminal GFP tag Over-expression of LipA and LipB with N-terminal myc tag Lamellipodin co-localised with L. monocytogenes in Hela M cells Staining of InlA(pNF8) infected Hela M cells with anti-lpd antibody Over-expression of Lpd-EGFP in Hela M cells infected with L. monocytogenes InlA(pJEBAN6) Discussion The visualisation of L. monocytogenes Comparison of the three methods used in visualisation of L. monocytogenes Analysis of the chromosomal and plasmid expression of mcherry The distribution of PIPs during L. monocytogenes infection PI4P, PI(4,5)P 2, PI(3,4,5)P PI3P PI(3,4)P 2 and lamellipodin Akt-PH recognition of PI(3,4)P 2 and PI(3,4,5)P Discussion for the expression of EGFP/YFP tagged PIP-binding domains The localisation of LipA and LipB Chapter 6 General discussion References Word count: 49,161 [5] List of Figures Figure 1.1 Different stages in the life cycle of L. monocytogenes...25 Figure 1.2 Cytoskeletal rearrangements induced during L. monocytogenes invasion of host cells...28 Figure 1.3 Schematic representation of the structural features of the 25 members of the internalin family encoded within the L. monocytogenes EGDe genome.31 Figure 1.4 a) Domain organisation of the ActA protein, its presentation on the bacterium, and the target sites for host cell proteins. b) Model of actin assembly induced by ActA...40 Figure 1.5 The PrfA regulon in L. monocytogenes..42 Figure 1.6 Model of bacterial dissemination from intestinal villi to liver and mesenteric lymph nodes.46 Figure 1.7 Successive steps of human listeriosis.47 Figure 1.8 The activation of macrophages 50 Figure 1.9 a) The structure of PI. b) Metabolic reactions leading to the generation of seven PIP species from PI. c) PIP-binding modules, their binding preference and some examples of proteins that contain them 57 Figure 1.10 Diagrammatic representation of PIP metabolism and actin recruitment.59 Figure 1.11 Pathogen trafficking pathways and host phosphoinositide metabolism 65 Figure 1.12 ClustalW (1.83) multiple protein sequence alignment of LipA, LipB in L. monocytogenes with the lipid phosphatase myotubularin in human (MTM1) and Caenorhabditis elegans Figure 2.1 Schematic picture of site-directed mutagenesis..86 Figure 2.2 a) Schematic picture of the generation of a knock out plasmid. b) Schematic picture of mutagenesis by homologues recombination 88 Figure 3.1 Intracellular survival assay of L. monocytogenes EGDe in Hela M cells at MOI 50 and Figure 3.2 Intracellular survival assay of L. monocytogenes EGDe in Hela M cells at MOI 1 and Figure 3.3 Intracellular survival assay on Hela M cells with L. monocytogenes EGDe, EGDe::ΔlipA, EGDe::ΔlipB and EGDe::Δhly at MOI of [6] Figure 4.1 Intracellular survival assay on Hela M cells with L. monocytogenes EGDe and InlA at MOI Figure 4.2 Confirmation of paula-lipb and paula-plcb by colony PCR Figure 4.3 Confirmation of the InlA::ΔlipA, InlA::ΔlipB, InlA::ΔlipAΔlipB, InlA::Δhly, InlA::ΔplcB and InlA::ΔhlyΔplcB mutations by colony PCR 106 Figure 4.4 Intracellular survival assay on Caco-2 cells with L. monocytogenes InlA, InlA::ΔlipA and InlA::Δhly at MOI Figure 4.5 Confirmation of punk1-lipa plasmid by colony PCR with punk1-f/r primers 110 Figure 4.6 punk1 plasmid stability test 110 Figure 4.7 Complementation of lipa with punk1-lipa plasmid in Caco-2 cells at MOI Figure 4.8 Intracellular survival assay on Hela M cells with L. monocytogenes InlA, InlA::ΔplcB and InlA::ΔhlyΔplcB at MOI Figure 4.9 Intracellular survival assay on Hela M cells with L. monocytogenes InlA, InlA::ΔplcA, InlA::ΔplcB and InlA::ΔlipAΔlipB at MOI of Figure 4.10 a) Confirmation of punk1-lipb by colony PCR with punk1-f/r primers. b) Confirmation of punk1-lipb by Pst I/Sac I diegestion. c) Schematic diagram of the Pst I and Sac I restriction site on punk1-lipb.115 Figure 4.11 Complementation of lipb with punk1-lipb plasmid in Hela M cell at MOI Figure 4.12 Intracellular survival assay on J774.A1 cells with L. monocytogenes InlA, InlA::ΔplcA, InlA::ΔplcB and InlA::ΔlipAΔlipB at MOI Figure 4.13 Phalloidin staining of Hela M cells infected with InlA, InlA::ΔlipA, InlA::ΔlipB, InlA::ΔhlyΔplcB and uninfected control 118 Figure 4.14 The percentage of L. monocytogenes associated with actin or comet tails and the number of bacteria per cell 4.5 hr post-infection Figure 4.15 Photos of the plaque assay on Hela M cells Figure 4.16 Number of plaques formed by L. monocytogenes in plaque assay on Hela M cells 122 Figure 4.17 Plasmid complementation of lipa in plaque assay 123 Figure 4.18 Internalisation of InlA, InlA::ΔlipA, InlA::ΔlipB and InlA::ΔlipAΔlipB into Hela M cells at MOI [7] Figure 5.1 Schematic diagram of the strategy used for constructing chromosomal fusion of mcherry or GFP uv in L. monocytogenes Figure 5.2 a) Confirmation of pbluescript-rpond-gfp uv /mcherry by colony PCR with T3 and T7 primers. b) Confirmation of paula-gfp uv /mcherry with M13-F/R primers Figure 5.3 a) Confirmation of InlA, InlA::GFP uv and InlA::mCherry by colony PCR. b) Schematic diagram of the primer binding sites at the GFPuv or mcherry insertion region on the L. monocytogenes chromosome..134 Figure 5.4 Growth curve of L. monocytogenes InlA and InlA::GFPuv in TSB media..135 Figure 5.5 Intracellular survival assay in Hela M cells with L. monocytogenes InlA and InlA::GFP uv at MOI Figure 5.6 Visualisation of InlA::GFP uv (left) and InlA (right) with confocal microscope.137 Figure 5.7 Schematic diagram of the strategy used for constructing the punk1-prpob-mcherry plasmid..139 Figure 5.8 a) Confirmation of punk1-mcherry by colony PCR with punk1-f/r primers. b) Confirmation of the mcherry orientation in punk1-mcherry plasmid by EcoR I/Nco I digestion. c) Confirmation of punk1-prpob-mcherry with punk1-f/r primers Figure 5.9 Intracellular survival assay in Hela M cells with L. monocytogenes InlA, InlA(pJEBAN6) and InlA(pNF8) at MOI Figure 5.10 Visualisation of the mid-log phase InlA(pJEBAN6) and InlA(pNF8) by fluorescent microscope..144 Figure 5.11 Imaging of InlA(pJEBAN6) and InlA(pNF8) in Hela M cells 4 hr post-infection.145 Figure 5.12 InlA(pJEBAN6) and InlA(pNF8) infected Hela M cells 24 hr post-infection.146 Figure 5.13 Staining of L. monocytogenes infected Hela M cell with anti-listeria antibody..147 Figure 5.14 Hela M cells expressing EGFP-FENS/FYVE. b) EGFP-FENS/FYVE co-localised with L. monocytogenes InlA in Hela M cells at 1.5 hr post-infection Figure 5.15 a) Hela M cells expressing EGFP-FAPP1-PH. b) Localisation of EGFP-FAPP1-PH in Hela M cells at 1.5 hr post-infection by L. monocytogenes InlA.152 [8] Figure 5.16 a) Hela M cells expressing EGFP-PLCδ-PH. b) Localisation of EGFP-PLCδ-PH in Hela M cells at 2.5 hr post-infection by L. monocytogenes InlA Figure 5.17 a) Hela M cells expressing YFP-ARNO-PH. b) Localisation of YFP-ARNO-PH in Hela M cells at 3.5 hr post-infection by L. monocytogenes InlA Figure 5.18 a) Hela M cells expressing EGFP-TAPP1-PH. b) EGFP-TAPP1-PH co-localised with L. monocytogenes InlA in Hela M cells at 6 hr post-infection. c) EGFP-TAPP1-PH co-localised with L. monocytogenes InlA in Hela M cells at 24 hr post-infection.156 Figure 5.19 a) Hela M cells expressing YFP-Akt-PH. b) Localisation of YFP-Akt-PH in Hela M cells at 3.5 hr post-infection by L. monocytogenes InlA..157 Figure 5.20 Localisation of PI(3,4)P 2 with InlA::ΔlipA, InlA::ΔlipB and InlA::ΔlipAΔlipB in Hela M cells Figure 5.21 Percentage of L. monocytogenes InlA and mutant strains co-localised with actin or PI(3,4)P 2 in Hela M cells 6 hr post-infection Figure 5.22 a) Colony PCR of pegfp-lipa with lipaexpr-f/r primers. b) Colony PCR of pegfp-lipb with LipBexpr-F/R primers.162 Figure 5.23 Over-expression of EGFP, LipA-EGFP and LipB-EGFP in Hela M cells 164 Figure 5.24 Co-localisation of LipB-EGFP with anti-golgi markers in Hela M cells 165 Figure 5.25 Over-expression of neutralised LipB in Hela M cells..166 Figure 5.26 Over-expression of myc-lipa and myc-lipb in Hela M cells.168 Figure 5.27 Anti-lamellipodin and phalloidin staining of Hela M cells..170 Figure 5.28 Co-localisation of L. monocytogenes InlA(pNF8) with lamellipodin and actin at 4 hr post-infection in Hela M cells 171 Figure 5.29 Co-localisation of L. monocytogenes InlA(pNF8) with lamellipodin and actin at 8 hr post-infection in Hela M cells 172 Figure 5.30 Co-localisation of L. monocytogenes InlA(pNF8) with lamellipodin and actin at 8 hr post-infection in Hela M cells 173 Figure 5.31 Over-expression of Lpd-EGFP in Hela M cells 175 Figure 5.32 Co-localisation of L. monocytogenes InlA(pJEBAN6) with Lpd-EGFP at 4 hr post-infection in Hela M cells Figure 5.33 Co-localisation of L. monocytogenes InlA(pJEBAN6) with Lpd-EGFP at 8 hr [9] post-infection in Hela M cells Figure 5.34 Co-localisation of L. monocytogenes InlA(pJEBAN6) with Lpd-EGFP at 8 hr post-infection in Hela M cells Figure 5.35 A hypothesized role of LipA during L. monocytogenes infection.189 Figure 6.1 Hypothesized actin- and lamellipodin-based intracellular movement cell-to-cell spread by L. monocytogenes..195 [10] List of Tables Table 2.1 Bacteria strains, features and sources involved in this study...73 Table 2.2 Plasmids, features and sources used in this study 74 Table 2.3 List of primers used in this study 80 Table 4.1 The 5 and 3 fragments of the target genes cloned in the construction of the deletion plasmids Table 5.1 The fluorescence level of InlA and InlA::GFP uv overnight cultures Table 5.2 Fluorescence level of mcherry expressed from punk1-prpob-mcherry in E. coli and L. monocytogenes..141 Table 5.3 Fluorescence level of DsRedExpress or GFP mut1 expressed from pjeban6 and pnf8 in E. coli and L. monocytogenes..143 Table 5.4 Erm concentration affected the percentage of InlA containing pjeban Table 5.5 The six probes used in this study for the recognition of PIPs 148 Table 5.6 Codon usage of mcherry, DsRedExpress and GFP mut1 by L. monocytogenes and E. coli..184 [11] Abstract Submitted by JIAHUI WANG for the Degree of Doctor of Philosophy in The University of Manchester and entitled Modulation of Phosphoinositide Metabolism by Intracellular Pathogenic Bacteria Listeria monocytogenes in April, Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a wide ecological niche and causes a number of diseases in human and animals. It invades mammalian host cells and escapes from the vacuoles prior to replication in the host cell cytoplasm and infecting adjacent cells via actin-based mobility. Phosphoinositide (PIP) metabolism is essential to mammalian cells in signal transduction, actin remodelling, endosome dynamics and membrane trafficking. Modulation of host PIP metabolism by bacteria PIP phosphatases is important for pathogenicity and virulence of many human pathogens. In this study the function of two L. monocytogenes tyrosine and inositol phosphatases LipA and LipB were studied in vitro. The lipa and lipb deletion mutants generated in EGDe and InlA strains were not affected in invasion but were attenuated in intracellular growth in Caco-2 and Hela M cell lines but not in mouse macrophages. Deletion of lipa or lipb did not affect the actin polymerisation but caused reduced plaque number in the plaque assay. The turnover of five PIPs in Hela M cells during L. monocytogenes infection were studied by expression of fluorescent protein tagged domains that specifically recognizes individual PIPs. L. monocytognenes did not affect the metabolism of PI4P, PI(4,5)P 2, PI(3,4,5)P 3 but co-localised with PI3P at 1.5 hr post-infection and with PI(3,4)P 2 at 6 hr to 24 hr post-infection. The PI(3,4)P 2 effector protein lamellipodin was discovered to be recruited to actin-associated L. monocytogenes at 4 hr to 24 hr post-infection in Hela M cells. This discovery leads to the hypothesis of a novel mechanism of lamellipodin-dependant cell-to-cell spread. The lipa mutant was found to be attenuated in PI(3,4)P 2 recruitment and therefore hypothesized to participate in the proposed lamellipodin pathway by converting PI(3,5)P 2 into PI5P, leading to the activation of PI3K and subsequent production of PI(3,4)P 2. LipB showed partial localisation at the Golgi complex when over-expressed in Hela M cells, and it was assumed to act mainly as a protein-tyrosine phosphatase. In summary, this study provides some evidence on L. monocytogenes modulating host PIP metabolism by the production of inositol phosphatases. It gives us a better understanding on the intracellular growth of this pathogenic bacterium, and on the interaction between host and parasite. [12] Declaration No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. Copyright Statement The author of this thesis (including any appendices and/or schedules to this thesis) owns the copyright or related rights in it and she has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property and any reproductions of copyright works
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