The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation
Publication Date
April 26, 2011
Journal
PLOS ONE
Authors
Heather Piscatelli, Shalaka A. Kotkar, Megan E. Mc Bee, Sureshkumar Muthupalani, et al
Volume
6
Issue
4
Pages
e19331
DOI
https://dx.plos.org/10.1371/journal.pone.0019331
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0019331
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/21541301
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082576
Europe PMC
http://europepmc.org/abstract/MED/21541301
Web of Science
000290018400053
Scopus
79955604109
Mendeley
http://www.mendeley.com/research/ehec-type-iii-effector-nlel-e3-ubiquitin-ligase-modulates-pedestal-formation
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Mendeley 3223 Apr 18:08 UTC

Mendeley | Further Information

{"title"=>"The ehec type iii effector nlel is an e3 ubiquitin ligase that modulates pedestal formation", "type"=>"journal", "authors"=>[{"first_name"=>"Heather", "last_name"=>"Piscatelli", "scopus_author_id"=>"37115873100"}, {"first_name"=>"Shalaka A.", "last_name"=>"Kotkar", "scopus_author_id"=>"38461330600"}, {"first_name"=>"Megan E.", "last_name"=>"McBee", "scopus_author_id"=>"57193788081"}, {"first_name"=>"Sureshkumar", "last_name"=>"Muthupalani", "scopus_author_id"=>"26534624500"}, {"first_name"=>"David B.", "last_name"=>"Schauer", "scopus_author_id"=>"7005864970"}, {"first_name"=>"Robert E.", "last_name"=>"Mandrell", "scopus_author_id"=>"7006646680"}, {"first_name"=>"John M.", "last_name"=>"Leong", "scopus_author_id"=>"7102532613"}, {"first_name"=>"Daoguo", "last_name"=>"Zhou", "scopus_author_id"=>"7403394523"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-79955604109", "doi"=>"10.1371/journal.pone.0019331", "pui"=>"361702820", "issn"=>"19326203", "pmid"=>"21541301", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "sgr"=>"79955604109"}, "id"=>"368e9a79-f7e1-3d4b-b60f-ab5f9ebecf93", "abstract"=>"Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.", "link"=>"http://www.mendeley.com/research/ehec-type-iii-effector-nlel-e3-ubiquitin-ligase-modulates-pedestal-formation", "reader_count"=>31, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Librarian"=>1, "Researcher"=>8, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Student > Master"=>1, "Student > Bachelor"=>6, "Lecturer"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Librarian"=>1, "Researcher"=>8, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Student > Master"=>1, "Student > Bachelor"=>6, "Lecturer"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>21, "Medicine and Dentistry"=>2, "Business, Management and Accounting"=>1, "Chemistry"=>2, "Social Sciences"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>2}, "Social Sciences"=>{"Social Sciences"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>21}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}}, "reader_count_by_country"=>{"Canada"=>1, "United Kingdom"=>1}, "group_count"=>1}
Scopus 3107 Feb 12:16 UTC

Scopus | Further Information

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Figshare09 Jan 22:01 UTC

Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/779945"], "description"=>"<p>(<b>A</b>) NleL-mediated self-ubiquitination requires ATP, ubiquitin, E1 and E2. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL<sup>59–782</sup> were incubated at 35°C for 90 min, and the Western blot was performed using polyclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). (<b>B</b>) NleL C753 residue is required for its E3 ubiquitin ligase activity. Reactions containing ubiquitin, E1 and UbcH5a were incubated with the wild type GST-NleL, GST-NleL<sup>C688S</sup> or GST-NleL<sup>C753S</sup> or GST-NleL<sup>C753A</sup> at 35°C for 90 min. The Western blot was performed using monoclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom).</p>", "links"=>[], "tags"=>["Infectious diseases", "microbiology"], "article_id"=>450308, "categories"=>["Microbiology", "Infectious Diseases"], "users"=>["Heather Piscatelli", "Shalaka A. Kotkar", "Megan E. McBee", "Sureshkumar Muthupalani", "David B. Schauer", "Robert E. Mandrell", "John M. Leong", "Daoguo Zhou"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019331.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Self_ubiquitination_of_GST_NleL_/450308", "title"=>"Self-ubiquitination of GST-NleL.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:51:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/780027"], "description"=>"<p>(<b>A</b>) The expression and secretion levels of Tir in either the wild-type EHEC or the mutant EHEC expressing the chromosomal catalytically-dead mutant NleL<sup>C753A</sup> was examined by Western blot. (<b>B</b>) Intracellular cAMP levels are an indication of the translocation of CyaA fusion proteins in EHEC WT (ZP250) and EHEC TTSS deficient mutant (<i>escF</i>; ZP2251) strains. Cells were infected for 4 hrs, and the adenylate cyclase activity was determined. The data were from three independent experiments, with standard deviations shown as error bars. cAMP values are presented as pmol per milligram of total cellular protein. The expression levels of the NleL-CyaA fusions were found to be similar by Western blot as shown on the bottom panel.</p>", "links"=>[], "tags"=>["tir", "translocation", "nlel", "hela"], "article_id"=>450387, "categories"=>["Microbiology", "Infectious Diseases"], "users"=>["Heather Piscatelli", "Shalaka A. Kotkar", "Megan E. McBee", "Sureshkumar Muthupalani", "David B. Schauer", "Robert E. Mandrell", "John M. Leong", "Daoguo Zhou"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019331.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Secretion_of_Tir_and_translocation_of_NleL_into_HeLa_cells_/450387", "title"=>"Secretion of Tir and translocation of NleL into HeLa cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:52:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/780144"], "description"=>"<p>HeLa cells were infected at a multiplicity of infection of 100 with wild type EHEC, <i>nleL<sup>C753A</sup></i>, or <i>nleL<sup>C753A</sup></i> harboring plasmid expressing wild-type NleL. Cells were infected for 6 hours. (<b>A</b>) Bacteria were visualized by staining with an anti-EHEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (<b>B</b>) The number of micro-clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–4 pedestals, 5–10 pedestals and >10 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars.</p>", "links"=>[], "tags"=>["ligase", "nlel", "modulating", "ehec", "pedestal"], "article_id"=>450505, "categories"=>["Microbiology", "Infectious Diseases"], "users"=>["Heather Piscatelli", "Shalaka A. Kotkar", "Megan E. McBee", "Sureshkumar Muthupalani", "David B. Schauer", "Robert E. Mandrell", "John M. Leong", "Daoguo Zhou"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019331.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_E3_ligase_activity_of_NleL_is_involved_in_modulating_EHEC_pedestal_formation_/450505", "title"=>"E3 ligase activity of NleL is involved in modulating EHEC pedestal formation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:52:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/780263"], "description"=>"<p>HeLa cells were infected at a multiplicity of infection of 100 with wild type EPEC, or EPEC harboring plasmid expressing wild-type EHEC NleL or NleL<sup>C753A</sup>. Cells were infected for 4 hours. (<b>A</b>) Bacteria were visualized by staining with an anti-EPEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (<b>B</b>) The number of micro-clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–10 pedestals, 11–20 pedestals and >20 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars.</p>", "links"=>[], "tags"=>["e3", "ligase", "ehec", "nlel", "modulates", "epec", "pedestal"], "article_id"=>450623, "categories"=>["Microbiology", "Infectious Diseases"], "users"=>["Heather Piscatelli", "Shalaka A. Kotkar", "Megan E. McBee", "Sureshkumar Muthupalani", "David B. Schauer", "Robert E. Mandrell", "John M. Leong", "Daoguo Zhou"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019331.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_E3_ligase_activity_of_EHEC_NleL_down_modulates_EPEC_pedestal_formation_/450623", "title"=>"The E3 ligase activity of EHEC NleL down modulates EPEC pedestal formation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:53:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/780349"], "description"=>"<p>(<b>A</b>) <i>C. rodentium</i> NleL is an E3 ubiquitin ligase. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL or GST-NleL<sup>C753S</sup> were incubated at 35°C for 90 min, and the Western blot was performed using polyclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). (<b>B)</b> Fecal bacteria shedding of the wild-type strain (DBS130), the <i>nleL</i> null mutant strain, the <i>nleL</i> mutant strain expressing the wild-type NleL, or the <i>nleL</i> mutant strain complemented with the catalytically-dead NleL<sup>C753S</sup> in <i>C. rodentium</i>. (<b>C</b>) Virulence of <i>C. rodentium</i> strains as indicated by weight of the mice post-inoculation. The percent weight change of the mice over the 13 days post-inoculation was measured. The error bars indicate standard errors. Mice infected with <i>nleL</i> deletion strain (DBS792) had significantly reduced weight loss when compared to the wild type (DBS130). This could be partially rescued by plasmid expressing the full length NleL (DBS793) but not by plasmid expressing the catalytically inactive NleL<sup>C753S</sup> mutant (DBS794). (<b>D</b>) Contribution of <i>nleL</i> to mouse colitis caused by <i>C. rodentium</i>. Pathology of mouse colon by different <i>C. rodentium</i> strains, as indicated by histologic activity index (sum of lesion scores). At day 13 post-inoculation colon tissue was scored for lesions: inflammation, edema, epithelial defects, crypt atrophy, hyperplasia, and dysplasia. Mice infected with <i>nleL</i> deletion strain showed significantly reduced lesions when compared to the wild type (P<0.05). This could be rescued by plasmid expressing the full length NleL but not by plasmid expressing the catalytically inactive NleL<sup>C753S</sup> mutant (P<0.01).</p>", "links"=>[], "tags"=>["Infectious diseases", "microbiology"], "article_id"=>450714, "categories"=>["Microbiology", "Infectious Diseases"], "users"=>["Heather Piscatelli", "Shalaka A. Kotkar", "Megan E. McBee", "Sureshkumar Muthupalani", "David B. Schauer", "Robert E. Mandrell", "John M. Leong", "Daoguo Zhou"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019331.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Contribution_of_nleL_to_C_rodentium_virulence_/450714", "title"=>"Contribution of <i>nleL</i> to <i>C. rodentium</i> virulence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:54:03"}
PMC Usage Stats 129317 Feb 15:56 UTC

PMC Usage Stats | Further Information

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Relative Metric 33087223 Feb 18:42 UTC

Relative Metric

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