Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection
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{"title"=>"Visual genome-wide RNAi screening to identify human host factors required for Trypanosoma cruzi infection", "type"=>"journal", "authors"=>[{"first_name"=>"Auguste", "last_name"=>"Genovesio", "scopus_author_id"=>"55932204200"}, {"first_name"=>"Miriam A.", "last_name"=>"Giardini", "scopus_author_id"=>"12792581900"}, {"first_name"=>"Yong Jun", "last_name"=>"Kwon", "scopus_author_id"=>"35080680400"}, {"first_name"=>"Fernando", "last_name"=>"de Macedo Dossin", "scopus_author_id"=>"37861415900"}, {"first_name"=>"Seo Yeon", "last_name"=>"Choi", "scopus_author_id"=>"40561049300"}, {"first_name"=>"Nam Youl", "last_name"=>"Kim", "scopus_author_id"=>"40561426100"}, {"first_name"=>"Hi Chul", "last_name"=>"Kim", "scopus_author_id"=>"35080518300"}, {"first_name"=>"Sung Yong", "last_name"=>"Jung", "scopus_author_id"=>"55471817500"}, {"first_name"=>"Sergio", "last_name"=>"Schenkman", "scopus_author_id"=>"7005825932"}, {"first_name"=>"Igor C.", "last_name"=>"Almeida", "scopus_author_id"=>"26643110500"}, {"first_name"=>"Neil", "last_name"=>"Emans", "scopus_author_id"=>"6701362010"}, {"first_name"=>"Lucio H.", "last_name"=>"Freitas-Junior", "scopus_author_id"=>"6507355684"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"79956326721", "doi"=>"10.1371/journal.pone.0019733", "pui"=>"361803802", "pmid"=>"21625474", "scopus"=>"2-s2.0-79956326721", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)"}, "id"=>"0ec79bad-fad7-35bf-85c3-648ea6416ef7", "abstract"=>"The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.", "link"=>"http://www.mendeley.com/research/visual-genomewide-rnai-screening-identify-human-host-factors-required-trypanosoma-cruzi-infection-6", "reader_count"=>11, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>1, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>1, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>6, "Medicine and Dentistry"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>6}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Colombia"=>1, "Mexico"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/388003", "https://ndownloader.figshare.com/files/388024", "https://ndownloader.figshare.com/files/388119"], "description"=>"<div><p>The protozoan parasite <em>Trypanosoma cruzi</em> is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in <em>T. cruzi</em> infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for <em>T. cruzi</em> infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of <em>T. cruzi</em> infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.</p> </div>", "links"=>[], "tags"=>["genome-wide", "rnai", "factors"], "article_id"=>136621, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.s001", "https://dx.doi.org/10.1371/journal.pone.0019733.s002", "https://dx.doi.org/10.1371/journal.pone.0019733.s003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Visual_Genome_Wide_RNAi_Screening_to_Identify_Human_Host_Factors_Required_for_Trypanosoma_cruzi_Infection/136621", "title"=>"Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for <em>Trypanosoma cruzi</em> Infection", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-05-20 01:50:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/772834"], "description"=>"<p>U2OS cells were seeded onto reverse transfection arrays comprising 250–300 µm spots spaced center-to-center at 500 µm intervals on a glass wafer. Spots contained p65 specific or control siRNAs (scrambled siRNA) and a red fluorescent siRNA in an encapsulation mixture (siGLO Red). Cells were incubated for 48 hours and then fixed and stained with anti-p65 antibodies. Confocal images were acquired 48 hours post-transfection. Scrambled siRNA was chosen as a negative control because it does not target any gene in the human genome. (<b>A</b>) Anti-p65 labeling of two p65 and two scrambled siRNA spots. (<b>B</b>) Spot images labeled with siGLO Red. (<b>C</b>) DNA staining (DRAQ5) showing an equal distribution of cells. (<b>D</b>) Overlay image. Scale bar represents 250 µm. (<b>E</b>) Quantification of p65 silencing using MetaXpress software (Molecular Devices). Plot showing p65 labeling (intensity/pixel/cell) for cells outside spots or overlaid on scrambled siRNA or p65 siRNA containing spots. ***p<0.0001 (unpaired t-test), n = 8.</p>", "links"=>[], "tags"=>["microarray"], "article_id"=>443193, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Proof_of_concept_of_the_microarray_screening_/443193", "title"=>"Proof-of-concept of the microarray screening.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-05-20 00:53:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/772946"], "description"=>"<p>Schematic illustration of how the developed software recognized the siRNA spot, the cells and the parasites for subsequent measurements. (<b>A</b>) All images of the <i>T. cruzi</i> primary screening were acquired in two different channels, as shown in (<b>B</b>) and (<b>D</b>). (<b>B</b> and <b>C</b>) Optically addressable siRNA spots were labeled with siGLO Red to enable their localization on the arrays. (<b>D</b>) Cell and parasite nuclei were stained using DRAQ5. As the raw images were acquired in a single wavelength, cells and parasites were artificially separated and emphasized in different colors (<b>E</b> and <b>F</b>, respectively), generating artificial two-wavelength images (<b>G</b>). This allowed for individual cell detection and quantification of several independent descriptors to measure infection (<b>H</b>). An enlarged panel showing cell detection by the software in greater detail is in the bottom left. The cell nuclei and boundaries are outlined in white and yellow/green, respectively (arbitrary colors). Scale bar represents 150 µm.</p>", "links"=>[], "tags"=>["genetics and genomics", "microbiology", "Computational biology"], "article_id"=>443308, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Microarray_screening_image_analysis_/443308", "title"=>"Microarray screening image analysis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-05-20 00:55:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/773101"], "description"=>"<p>Examples of data from the primary screening performed over microarrays showing that the knockdown of some genes inhibited infection by <i>T. cruzi</i> parasites. In (<b>A</b>) and (<b>B</b>), two examples are shown of genes selected as hits: PCDHA13 (protocadherin alpha 13) and C20orf200 (chromosome 20 open reading frame 200), respectively. Scrambled siRNA (<b>C</b>) was used as a negative control and p65 siRNA (<b>D</b>) was used as a transfection control (see Materials and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019733#s2\" target=\"_blank\">Methods</a>). Cells and parasites are pseudocolored in white, while siRNA spots are pseudocolored in red. The yellow dashed boxes outline the spot region. Cells within the spot were silenced for the indicated genes and compared with the cells lying outside the spot for their infection ratio and parasite load through software analysis. Scale bar represents 80 µm.</p>", "links"=>[], "tags"=>["microarray"], "article_id"=>443463, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Genome_wide_microarray_screening_results_/443463", "title"=>"Genome-wide microarray screening results.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-05-20 00:57:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/773266"], "description"=>"<p>The secondary screening was performed in 96-well plates. To validate this format, U2OS cells were seeded, transfected with the appropriate siRNAs and then infected with <i>T. cruzi</i> trypomastigotes as detailed in Materials and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019733#s2\" target=\"_blank\">Methods</a>. From left to right, cells were immunostained for p65 protein in the absence of <i>T. cruzi</i> parasites, in the absence of siRNAs, in the presence of scrambled siRNA or in the presence of p65 siRNA, respectively. (<b>A</b>) Immunofluorescence staining against p65 protein (green). (<b>B</b>) DNA staining of cell and parasite nuclei (DRAQ5, purple). (<b>C</b>) Overlay images. In the bottom panel, a wide view of the plate wells, showing p65 protein labeling (green) and cells (white). The rightmost column evidences the p65 knockdown when cells were transfected with p65 siRNA. Scale bar represents 80 µm.</p>", "links"=>[], "tags"=>["genetics and genomics", "microbiology", "Computational biology"], "article_id"=>443621, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Validation_of_the_secondary_screening_/443621", "title"=>"Validation of the secondary screening.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-05-20 01:00:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/773407"], "description"=>"<p>To confirm specificity and exclude off-target effects of the siRNA pools used in the primary and secondary screens, the 4 different siRNA duplexes from the pool targeting each gene were separated and individually transfected to U2OS cells in 96-well plates following the same experimental conditions used for secondary screen. (<b>A</b>) Schematic representation of the four siRNAs targeting the calcium binding protein 2 (CABP2) mRNA sequence. bp = base pairs. (<b>B</b>) Pictures showing CABP2 gene silencing using the four different siRNAs depicted in (<b>A</b>). U2OS cells were transfected with each one of the siRNAs and then infected with <i>T. cruzi</i> parasites. Pictures <b>a, b, c</b> and <b>d</b> show a decrease in infection when compared to scrambled siRNA (bottom picture). All 13 genes tested were confirmed by at least two individual siRNAs, demonstrating that the infection inhibition seen in primary and secondary screens was not due to an off target effect. Scale bar represents 80 µm.</p>", "links"=>[], "tags"=>["specificity", "sirna"], "article_id"=>443762, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Confirming_specificity_of_siRNA_silencing_/443762", "title"=>"Confirming specificity of siRNA silencing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-05-20 01:02:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/773507"], "description"=>"<p>U2OS cells were transfected with the siRNAs (scrambled siRNA, CDH11, CHP, FUT8 and NICE-3) and total RNA was isolated from each sample after a 48-hour incubation. cDNAs were prepared and endogenous mRNA levels were measured using real-time PCR. The relative copy numbers of the transcripts were normalized to GAPDH and the knockdown was quantified using scrambled siRNA as the negative control.</p>", "links"=>[], "tags"=>["mrna", "levels", "quantitative", "Real-time"], "article_id"=>443863, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Checking_mRNA_levels_by_quantitative_real_time_PCR_/443863", "title"=>"Checking mRNA levels by quantitative real-time PCR.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-05-20 01:04:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/773571"], "description"=>"<p>*Data from Entrez Gene (<a href=\"http://www.ncbi.nlm.nih.gov/gene/\" target=\"_blank\">http://www.ncbi.nlm.nih.gov/gene/</a>).</p>#<p>Data from UniProtKB (<a href=\"http://www.uniprot.org/uniprot/\" target=\"_blank\">http://www.uniprot.org/uniprot/</a>).</p>", "links"=>[], "tags"=>["14", "genes"], "article_id"=>443930, "categories"=>["Biological Sciences", "Genetics", "Microbiology"], "users"=>["Auguste Genovesio", "Miriam A. Giardini", "Yong-Jun Kwon", "Fernando de Macedo Dossin", "Seo Yeon Choi", "Nam Youl Kim", "Hi Chul Kim", "Sung Yong Jung", "Sergio Schenkman", "Igor C. Almeida", "Neil Emans", "Lucio H. Freitas-Junior"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0019733.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_List_of_the_14_human_genes_that_are_important_for_T_cruzi_infection_/443930", "title"=>"List of the 14 human genes that are important for <i>T. cruzi</i> infection.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-05-20 01:05:30"}

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Relative Metric

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