Evidence for a Role of the Polysaccharide Capsule Transport Proteins in Pertussis Pathogenesis
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{"title"=>"Evidence for a role of the polysaccharide capsule transport proteins in pertussis pathogenesis", "type"=>"journal", "authors"=>[{"first_name"=>"Regina", "last_name"=>"Hoo", "scopus_author_id"=>"36025168700"}, {"first_name"=>"Jian Hang", "last_name"=>"Lam", "scopus_author_id"=>"56177599400"}, {"first_name"=>"Ludovic", "last_name"=>"Huot", "scopus_author_id"=>"6602743471"}, {"first_name"=>"Aakanksha", "last_name"=>"Pant", "scopus_author_id"=>"55695294000"}, {"first_name"=>"Rui", "last_name"=>"Li", "scopus_author_id"=>"57198938244"}, {"first_name"=>"David", "last_name"=>"Hot", "scopus_author_id"=>"6505943257"}, {"first_name"=>"Sylvie", "last_name"=>"Alonso", "scopus_author_id"=>"7102615191"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"25501560", "sgr"=>"84918840592", "doi"=>"10.1371/journal.pone.0115243", "scopus"=>"2-s2.0-84918840592", "pui"=>"600755189", "isbn"=>"1932-6203", "issn"=>"19326203"}, "id"=>"d0c0a6bf-e54d-36af-b1c9-eb643bb3c12f", "abstract"=>"Polysaccharide (PS) capsules are important virulence determinants for many bacterial pathogens. Bordetella pertussis, the agent of whooping cough, produces a surface associated microcapsule but its role in pertussis pathogenesis remained unknown. Here we showed that the B. pertussis capsule locus is expressed in vivo in murine lungs and that absence of the membrane-associated protein KpsT, involved in the transport of the PS polymers across the envelope, but not the surface-exposed PS capsule itself, affects drastically B. pertussis colonization efficacy in mice. Microarray analysis revealed that absence of KpsT in B. pertussis resulted in global down-regulation of gene expression including key virulence genes regulated by BvgA/S, the master two-component system. Using a BvgS phase-locked mutant, we demonstrated a functional link between KpsT and BvgA/S-mediated signal transduction. Whereas pull-down assays do not support physical interaction between BvgS sensor and any of the capsule locus encoded proteins, absence of KpsT impaired BvgS oligomerization, necessary for BvgS function. Furthermore, complementation studies indicated that instead of KpsT alone, the entire PS capsule transport machinery spanning the cell envelope likely plays a role in BvgS-mediated signal transduction. Our work thus provides the first experimental evidence of a role for a virulence-repressed gene in pertussis pathogenesis.", "link"=>"http://www.mendeley.com/research/evidence-role-polysaccharide-capsule-transport-proteins-pertussis-pathogenesis", "reader_count"=>24, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>2, "Student > Master"=>4, "Other"=>2, "Student > Bachelor"=>7}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>2, "Student > Master"=>4, "Other"=>2, "Student > Bachelor"=>7}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>4, "Nursing and Health Professions"=>1, "Materials Science"=>1, "Agricultural and Biological Sciences"=>11, "Medicine and Dentistry"=>1, "Sports and Recreations"=>1, "Chemistry"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Sports and Recreations"=>{"Sports and Recreations"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>11}, "Computer Science"=>{"Computer Science"=>1}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>1}}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1841646"], "description"=>"<p>(<b>A</b>) <b>Relative transcriptional activity of </b><b><i>vags</i></b><b>.</b> Total RNA was extracted from BPSM (solid bars), Δ<i>kpsT</i> (dotted bars) and Δ<i>kpsT</i>com (stripped bars) strains grown in virulent Bvg<sup>+</sup> phase. Real-time PCR analysis was performed using primers mapping in the <i>brkA</i>, <i>ptx</i>, <i>sphB1</i>, <i>fhaB</i>, <i>bvgA</i> and <i>bvgR</i> genes. <i>recA</i> gene was used as the endogenous control. Results are expressed as average relative quantification (RQ) vs wild type BPSM (RQ = 1). Results are representative of 3 independent experiments. (<b>B</b>) <b>Microarray analysis.</b> Total RNA was extracted from BPSM and Δ<i>kpsT</i> strains grown in virulent (Bvg<sup>+</sup>) phase. Microarray gene expression values were selected based on log<sub>2</sub> fold change <-0.8, with adjusted <i>p</i> value<0.01. Results are expressed as average fold change Δ<i>kpsT</i> compared to BPSM, negative value indicates gene repression. Solid bars represent standard deviation of 2 independent experiments.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269504, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g005", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Trancriptional_activity_in_kpsT_mutant_/1269504", "title"=>"Trancriptional activity in Δ<i>kpsT</i> mutant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841644"], "description"=>"<p>(<b>A</b>) <b>Detection of the polysaccharide capsule at the bacterial surface.</b> Mouse polyclonal anti-<i>Salmonella typhi</i> Vi antigen immune serum was co-incubated with non-permeabilized BPSM, KO<i>caps</i>, Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> bacteria strains as indicated in each flow cytometry plot. All bacteria strains were grown in Bvg<sup>-</sup> phase. Anti-mouse FITC-conjugated IgG was used as secondary antibody. Isotype-matched controls are incubated with an anti-mouse antibody as shown in black histogram. The fluorescent cells were detected by flow cytometry, with 20,000 events counted for each sample. A representative experiment is shown from three independent experiments, with percentage of fluorescent cells indicated in each panel. (<b>B</b>) <b>Lung colonization profiles.</b> Balb/C mice were infected intranasally with 5×10<sup>5</sup> CFU of <i>B. pertussis</i> BPSM, KO<i>caps</i>, Δ<i>kpsT</i>, Δ<i>kpsT</i>com, Δ<i>kpsE</i> and Δ<i>vipC</i> as indicated at each of graph plot. At the indicated time points, four infected mice per group were euthanized and their lungs were harvested, homogenized and plated on blood agar to determine the total number of CFU per lung. The results are expressed as the mean ± SEM of four mice per group. ** <i>p</i> value<0.01 and * <i>p</i> value<0.05 relative to BPSM. Results are representative of two independent experiments.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269502, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g003", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phenotypic_characterization_of_the_kpsT_kpsE_and_vipC_mutants_/1269502", "title"=>"Phenotypic characterization of the Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841640"], "description"=>"<p>Mice were infected intranasally with approx. 5×10<sup>6</sup> CFU of BPSM and the bacteria were recovered from the mice lungs at different time points (3 hours, day 3 and day 7 p.i.). Bacterial RNA extracted and purified from group of 4 mice were pooled and subjected to real-time PCR analysis using primers mapping in the <i>kpsT</i>, <i>vrg6</i>, <i>bvgA</i>, <i>fhaB</i> and <i>ptx</i> genes. <i>RecA</i> gene was used as the endogenous control. Results are expressed as the average RQ ± SD of triplicate versus BPSM inoculum. Dotted line represents RQ equal to 1 relative to BPSM inoculum. Mock-infected mice were used as negative control. The data obtained with two biological replicates are shown. Please refer to <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115243#pone.0115243.s001\" target=\"_blank\">S1 Table</a> for Pearson correlation scores between the two independent sets.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269498, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g001", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Relative_transcriptional_activity_of_vrgs_and_vags_in_BPSM_bacteria_recovered_from_mice_lungs_versus_in_vitro_BPSM_grown_in_virulent_phase_/1269498", "title"=>"Relative transcriptional activity of <i>vrgs</i> and <i>vags</i> in BPSM bacteria recovered from mice lungs versus <i>in vitro</i> BPSM grown in virulent phase.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841660"], "description"=>"<p>List of forward and reverse primers (from 5′ to 3′) used in Real-time PCR analysis.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269511, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.t003", "stats"=>{"downloads"=>2, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_List_of_forward_and_reverse_primers_from_5_8242_to_3_8242_used_in_Real_time_PCR_analysis_/1269511", "title"=>"List of forward and reverse primers (from 5′ to 3′) used in Real-time PCR analysis.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841661"], "description"=>"<div><p>Polysaccharide (PS) capsules are important virulence determinants for many bacterial pathogens. <i>Bordetella pertussis</i>, the agent of whooping cough, produces a surface associated microcapsule but its role in pertussis pathogenesis remained unknown. Here we showed that the <i>B. pertussis</i> capsule locus is expressed <i>in vivo</i> in murine lungs and that absence of the membrane-associated protein KpsT, involved in the transport of the PS polymers across the envelope, but not the surface-exposed PS capsule itself, affects drastically <i>B. pertussis</i> colonization efficacy in mice. Microarray analysis revealed that absence of KpsT in <i>B. pertussis</i> resulted in global down-regulation of gene expression including key virulence genes regulated by BvgA/S, the master two-component system. Using a BvgS phase-locked mutant, we demonstrated a functional link between KpsT and BvgA/S-mediated signal transduction. Whereas pull-down assays do not support physical interaction between BvgS sensor and any of the capsule locus encoded proteins, absence of KpsT impaired BvgS oligomerization, necessary for BvgS function. Furthermore, complementation studies indicated that instead of KpsT alone, the entire PS capsule transport machinery spanning the cell envelope likely plays a role in BvgS-mediated signal transduction. Our work thus provides the first experimental evidence of a role for a virulence-repressed gene in pertussis pathogenesis.</p></div>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269512, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243", "stats"=>{"downloads"=>4, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Evidence_for_a_Role_of_the_Polysaccharide_Capsule_Transport_Proteins_in_Pertussis_Pathogenesis_/1269512", "title"=>"Evidence for a Role of the Polysaccharide Capsule Transport Proteins in Pertussis Pathogenesis", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841659"], "description"=>"<p>Restriction sites are underlined.</p><p>List of forward and reverse primers used in cloning.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269510, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.t002", "stats"=>{"downloads"=>2, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_List_of_forward_and_reverse_primers_used_in_cloning_/1269510", "title"=>"List of forward and reverse primers used in cloning.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841652"], "description"=>"<p>(<b>A</b>) <b>Coomassie blue analysis.</b> 5 mg of solubilize cell lysate harvested from BPSM (untagged control) and BPSH was mixed with Ni-NTA agarose beads prior to loading onto a chromatography column. Lysate input, flow-through and batch eluted fractions were heated to 95°C for 15 min and analyzed under reducing 10% SDS-PAGE and stained with Coomassie blue. Lane IP; Input, FT; Flow through, E1; Eluted fraction 1, E2; Eluted fraction 2, E3; Eluted fraction 3, E4; Eluted fraction 4. Molecular weights are indicated on the right side. (<b>B</b>) <b>Western blot analysis of purified fractions with anti-His and anti-BvgS antibodies.</b> Lane IP; Input, FT; Flow through, E2; Eluted fraction 2, E3; Eluted fraction 3. Molecular weights are indicated on the right side. (<b>C</b>) <b>Detection of BvgS associated oligomers and BvgS monomer.</b> Purified His-BvgS from BPSH cells were mixed with equal volume of Laemmli SDS-PAGE buffer containing either no reducing agent (0% β-mercaptoethanol) or increasing concentrations (2.5% and 5% β-mercaptoethanol). The proteins samples were then subjected to either no heat or heat denaturation at 95°C for 15 min prior to SDS-PAGE analysis and Western blotted with anti-His or anti-BvgS antibody. Molecular weights are indicated on the right side. (<b>D</b>) <b>Detection of BvgS associated oligomers and BvgS monomer in BPSH and BPSH-KO</b><b><i>caps</i></b><b>.</b> Equal amount of purified His-BvgS from BPSH and BPSH-KO<i>caps</i> cells were mixed with Laemmli SDS-PAGE buffer containing either no reducing agent or with 5% β-mercaptoethanol. The proteins samples were then subjected to either no heat or heat denaturation at 95°C for 15 min. Equal amount of protein were loaded for each well for SDS-PAGE analysis and Western blotted with anti-BvgS antibody. Molecular weights are indicated on the right side. (<b>E</b>) <b>Detection of BvgS associated oligomers and BvgS monomer in BPSH, BPSH-Δ</b><b><i>kpsT</i></b><b> and BPSH-Δ</b><b><i>kpsT</i></b><b>com.</b> Equal amount of purified His-BvgS from BPSH, BPSH-Δ<i>kpsT</i> and BPSH-Δ<i>kpsT</i>com cells were mixed with Laemmli SDS-PAGE buffer containing either no reducing agent or with 5% β-mercaptoethanol. The proteins samples were then subjected to either no heat or heat denaturation at 95°C for 15 min. Equal amounts of protein were loaded for each well for SDS-PAGE analysis and Western blotted with anti-BvgS antibody. Molecular weights are indicated on the right side.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269507, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g007", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_and_purification_of_His_BvgS_from_B_pertussis_strains_/1269507", "title"=>"Expression and purification of His-BvgS from <i>B. pertussis</i> strains.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841642"], "description"=>"<p>(<b>A</b>) <b>Schematic organization of </b><b><i>B</i></b><b>. </b><b><i>pertussis</i></b><b> capsule operon.</b> The capsule operon of <i>B. pertussis</i> BPSM strain regulated under the capsule promoter is as shown in panel <b>a</b>, black cross represents mutational insertion found in the locus. Black, hashed and open arrows represent genes involved in polysaccharide transport, polysaccharide modification/translocation and polysaccharide biosynthesis respectively. Adapted from GeneDB. Dotted triangle in panel <b>b</b> (Δ<i>kpsT</i>), <b>c</b> (Δ<i>kpsE</i>) and <b>d</b> (Δ<i>vipC</i>) indicate site of deletion that render each mutant non-capsulated. The DIG-labeled probe binding region (black rounded arrow), restriction sites and size of restriction-digested chromosomal DNA for Southern blot analysis are as shown. (<b>B</b>) <b>Southern blot analysis.</b> Restriction-digested chromosomal DNA from BPSM and Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> were electrophoresed, transferred onto a nitrocellulose membrane and hybridized with the DIG-labeled probe (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115243#pone-0115243-g001\" target=\"_blank\">Fig. 1A</a> for probe binding site). Panel <b>a</b>, <i>EcoRI</i>-restricted BPSM and Δ<i>kpsT</i> DNA yielded 2.7-kb and 3.2-kb respectively. Panel <b>b</b>, Hind<i>III</i>-Nco<i>I</i> restricted BPSM and Δ<i>kpsE</i> DNA yielded 4.8-kb and 3.9-kb respectively. Panel <b>c</b>, Hind<i>III</i>-Nco<i>I</i> restricted BPSM and Δ<i>vipC</i> DNA yielded 4.8-kb and 3.4-kb respectively. (<b>C</b>) <b>Transcription efficacy of downstream gene.</b> Total RNA was extracted from exponential SS liquid cultures of BPSM, KO<i>caps</i>, Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> was reverse-transcribed followed by PCR amplification using primers specific to the endogenous control gene <i>recA</i>, and primers mapping to the respective downstream region of the deleted ORFs. The KO<i>caps</i> strain which was deleted for the entire capsule locus was used as a negative control. (<b>D</b>) <b>Growth kinetic profiles.</b> SS liquid medium was inoculated with BPSM (closed circles), Δ<i>kpsT</i> (open circles), Δ<i>kpsE</i> (closed triangles) and Δ<i>vipC</i> (open triangles) at initial OD<sub>600 nm</sub> of 0.5 at time-point 0 hour. OD<sub>600 nm</sub> was monitored for 52 hrs incubation at 37°C. The growth kinetics assay was performed twice independently for each strain and each culture condition. The data shown is representative of two independent experiments.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269500, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g002", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Construction_of_kpsT_kpsE_and_vipC_B_pertussis_mutants_/1269500", "title"=>"Construction of Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC B. pertussis</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841658"], "description"=>"<p><i>B. pertussis</i> strains used in this study.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269509, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.t001", "stats"=>{"downloads"=>2, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_B_pertussis_strains_used_in_this_study_/1269509", "title"=>"<i>B. pertussis</i> strains used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841657"], "description"=>"<p>Balb/C mice were infected intranasally with 5×10<sup>5</sup> CFU of <i>B. pertussis</i> BPSM (solid bars), KO<i>caps</i> (striped bars), KO<i>caps</i>:<i>kpsT</i> (dotted bars) and KO<i>caps</i>:<i>kpsMT</i> (open bars). At the indicated time points, four infected mice per group were euthanized and their lungs were harvested, homogenized and plated on blood agar to determine the total number of CFU per lung. The results are expressed as the mean ± SEM of four mice per group. ** <i>p</i> value<0.01 relative to KO<i>caps</i>. Results are representative of two independent experiments.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269508, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g008", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Lung_colonization_profile_of_B_pertussis_KO_caps_kpsT_and_KO_caps_kpsMT_strains_/1269508", "title"=>"Lung colonization profile of <i>B. pertussis</i> KO<i>caps</i>:<i>kpsT</i> and KO<i>caps</i>:<i>kpsMT</i> strains.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841647"], "description"=>"<p>BPSM, BvgS-VFT2, Δ<i>kpsT</i> and BvgS-VFT2-Δ<i>kpsT</i> strains were exponentially grown in virulent (Bvg<sup>+</sup>) and avirulent (Bvg<sup>-</sup>) phase. Western blot analysis was performed on whole cell extract (panel <b>a</b>) and 10x concentrated (panel <b>b</b>) or non-concentrated (panel <b>c</b>) culture supernatants using anti-BrkA (<b>a</b>), anti-PT (<b>b</b>) or anti-FHA (<b>c</b>) primary antibodies. Bars at the right of the blots represent protein densitometry quantification expressed as percentage of change relative to parental BPSM in Bvg<sup>+</sup> phase. Results are representative of three independent experiments. Molecular weights are indicated on the right side. (<b>B</b>) <b>Relative transcriptional activity of </b><b><i>vags</i></b><b>.</b> Total RNA was extracted from BPSM, BvgS-VFT2, Δ<i>kpsT</i> and BvgS-VFT2-Δ<i>kpsT</i> strains grown in virulent Bvg<sup>+</sup> phase. Real-time PCR analysis was performed using primers mapping in the <i>brkA</i>, <i>ptx</i>, and <i>sphB1</i> genes. <i>recA</i> gene was used as the endogenous control. Results are expressed for each target gene as average fold change ± SD of triplicate Ct values obtained with BvgS-VFT2, Δ<i>kpsT</i> and BvgS-VFT2-Δ<i>kpsT</i> versus the Ct value obtained with BPSM strain. The results are representative of two independent experiments. (<b>C</b>) <b>Lung colonization profile.</b> Balb/C mice were infected intranasally with 5×10<sup>5</sup> CFU of <i>B. pertussis</i> BvgS-VFT2, Δ<i>kpsT</i> and BvgS-VFT2-Δ<i>kpsT</i>. At the indicated time points, four infected mice per group were euthanized and their lungs were harvested, homogenized and plated on blood agar to determine the total number of CFU per lung. The results are expressed as the mean ± SEM of four mice per group. ** <i>p</i> value<0.01 relative to BPSM. Results are representative of two independent experiments.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269505, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g006", "stats"=>{"downloads"=>1, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Characterization_of_the_BvgS_VFT2_kpsT_mutant_A_Production_of_bvg_regulated_virulence_proteins_/1269505", "title"=>"Characterization of the BvgS-VFT2-Δ<i>kpsT</i> mutant. (A) Production of <i>bvg</i>-regulated virulence proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/1841645"], "description"=>"<p>BPSM, Δ<i>kpsT</i> and Δ<i>kpsT</i>com strains were exponentially grown in virulent Bvg<sup>+</sup> phase. Western blot analysis was performed on 10x concentrated or non-concentrated culture supernatants, and whole cell extracts using (<b>A</b>) anti-FHA, (<b>B</b>) anti-BrkA or (<b>C</b>) anti-PT primary antibodies. Bars at the bottom of each blot represent protein densitometry quantification expressed as percentage of change relative to parental BPSM and was determined from two independent western blot sets. Molecular weights are indicated on the right side. (<b>D</b>) SDS-PAGE and Coomassie blue staining of BPSM, Δ<i>kpsT</i> and Δ<i>kpsT</i>com whole cell lysate to estimate equal loading of protein content. Asterisks next to the Coomassie-stained bands indicate for loading control estimate. Bars represent protein densitometry quantification expressed as percentage of change relative to BPSM. Molecular weights are indicated on the left side.</p>", "links"=>[], "tags"=>["absence", "KpsT", "bvgs", "Pertussis Pathogenesis Polysaccharide", "pertussis pathogenesis", "PS capsule transport machinery", "pertussis colonization efficacy", "Polysaccharide Capsule Transport Proteins", "pertussis capsule locus", "role"], "article_id"=>1269503, "categories"=>["Biological Sciences"], "users"=>["Regina Hoo", "Jian Hang Lam", "Ludovic Huot", "Aakanksha Pant", "Rui Li", "David Hot", "Sylvie Alonso"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0115243.g004", "stats"=>{"downloads"=>1, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Production_of_bvg_regulated_virulence_proteins_in_kpsT_mutant_/1269503", "title"=>"Production of <i>bvg</i>-regulated virulence proteins in Δ<i>kpsT</i> mutant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-12 02:50:37"}

PMC Usage Stats | Further Information

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