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{"title"=>"Drosophila embryos as model systems for monitoring bacterial infection in real time", "type"=>"journal", "authors"=>[{"first_name"=>"Isabella", "last_name"=>"Vlisidou", "scopus_author_id"=>"10539638500"}, {"first_name"=>"Andrea J.", "last_name"=>"Dowling", "scopus_author_id"=>"7006290423"}, {"first_name"=>"Iwan R.", "last_name"=>"Evans", "scopus_author_id"=>"34874791600"}, {"first_name"=>"Nicholas", "last_name"=>"Waterfield", "scopus_author_id"=>"6601935218"}, {"first_name"=>"Richard H.", "last_name"=>"Ffrench-Constant", "scopus_author_id"=>"34975054200"}, {"first_name"=>"Will", "last_name"=>"Wood", "scopus_author_id"=>"56502476400"}], "year"=>2009, "source"=>"PLoS Pathogens", "identifiers"=>{"pui"=>"355237363", "issn"=>"15537366", "isbn"=>"1553-7374 (Electronic)", "doi"=>"10.1371/journal.ppat.1000518", "scopus"=>"2-s2.0-70049107898", "pmid"=>"19609447", "sgr"=>"70049107898"}, "id"=>"7aa33e07-674a-3c6d-abcc-466ba010c3ba", "abstract"=>"Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid 'freezing' phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.", "link"=>"http://www.mendeley.com/research/drosophila-embryos-model-systems-monitoring-bacterial-infection-real-time", "reader_count"=>85, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>4, "Researcher"=>33, "Student > Ph. D. Student"=>25, "Student > Postgraduate"=>1, "Student > Master"=>9, "Other"=>2, "Student > Bachelor"=>4, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>2, "Professor"=>3}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>4, "Researcher"=>33, "Student > Ph. D. Student"=>25, "Student > Postgraduate"=>1, "Student > Master"=>9, "Other"=>2, "Student > Bachelor"=>4, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>2, "Professor"=>3}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>10, "Agricultural and Biological Sciences"=>61, "Medicine and Dentistry"=>3, "Neuroscience"=>1, "Veterinary Science and Veterinary Medicine"=>1, "Social Sciences"=>1, "Computer Science"=>3, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Neuroscience"=>{"Neuroscience"=>1}, "Social Sciences"=>{"Social Sciences"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>61}, "Computer Science"=>{"Computer Science"=>3}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}, "Unspecified"=>{"Unspecified"=>2}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"Canada"=>1, "Netherlands"=>1, "United Kingdom"=>3, "France"=>1, "Chile"=>1, "Portugal"=>3, "Germany"=>1, "India"=>1, "Spain"=>1}, "group_count"=>2}

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• http://doi.org/10.1016/j.mib.2009.12.005
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• http://doi.org/10.1016/j.cub.2016.06.012

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/891479"], "description"=>"<p>(A) Stills from a movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s009\" target=\"_blank\">Video S9</a>) of hemocytes expressing <i>Rac^N17</i> following injection of Mcf1. Hemocytes expressing <i>Rac^N17</i> are localised at the anterior of the embryo and have decreased lamellipodia formation and movement compared to wild-type cells. However, despite these defects <i>Rac^N17</i> hemocytes fail to freeze after Mcf1 injection and continue to form small dynamic membrane ruffles (arrow) and filopodia (arrowheads). (B) Time-lapse movie stills (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s010\" target=\"_blank\">Video S10</a>) showing constitutively active <i>Rac^V12</i> expressing hemocytes following Mcf1 injection. <i>Rac^V12</i> expression in hemocytes causes reduced lamellipodia formation and migration when compared to wildtype cells. When exposed to Mcf1 these cells fail to display the freezing phenotype and like the <i>Rac^N17</i> expressing cells continue to make small dynamic protrusions (arrows). Scale bars represent 10 µm. Elapsed time is indicated in the upper right corner.</p>", "links"=>[], "tags"=>["defective", "rac", "evade", "mcf1", "mediated"], "article_id"=>561933, "categories"=>["Immunology", "Cell Biology", "Developmental Biology"], "users"=>["Isabella Vlisidou", "Andrea J. Dowling", "Iwan R. Evans", "Nicholas Waterfield", "Richard H. ffrench-Constant", "Will Wood"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000518.g006", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Embryos_with_defective_Rac_activity_evade_Mcf1_mediated_paralysis_/561933", "title"=>"Embryos with defective Rac activity evade Mcf1 mediated paralysis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-17 00:32:13"}
• {"files"=>["https://ndownloader.figshare.com/files/891345"], "description"=>"<p>(A) Stills from a timelapse movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s008\" target=\"_blank\">Video S8</a>) of hemocytes expressing <i>shibire^ts1</i> following injection with 0.2 µg µl⁻¹ Mcf1. Unlike wildtype cells, <i>shibire</i> mutant hemocytes do not freeze after exposure to Mcf1 and continue to migrate (asterisk) and extend large actin rich protrusions (arrows). Elapsed time is indicated in the upper right corner. (B) Injection of Alexa Fluro 555 labelled Mcf1 (Mcf1 555) into wildtype embryos shows localisation of labelled Mcf1 in hemocytes (white arrows) 1 hour after injection. Mcf1 can also be seen outside hemocytes indicating localisation in other surrounding cell types (arrowheads). Scale bars represent 10 µm.</p>", "links"=>[], "tags"=>["mcf1"], "article_id"=>561801, "categories"=>["Immunology", "Cell Biology", "Developmental Biology"], "users"=>["Isabella Vlisidou", "Andrea J. Dowling", "Iwan R. Evans", "Nicholas Waterfield", "Richard H. ffrench-Constant", "Will Wood"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000518.g005", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Internalisation_of_Mcf1_is_required_for_freezing_/561801", "title"=>"Internalisation of Mcf1 is required for freezing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-17 00:30:01"}
• {"files"=>["https://ndownloader.figshare.com/files/890776"], "description"=>"<p>(A) An embryo expressing GFP specifically in the hemocytes shows their characteristic pattern of distribution whereby the cells are arranged into three parallel lines running along the ventral surface of a stage 15 embryo (arrows). (B) Embryo injected with fluorescently labelled <i>E. coli</i> in the anterior region of the embryo (asterisk). 20 minutes after injection hemocytes become labelled as they bind the injected bacteria (arrows). (C) Hemocytes within <i>dscam⁰⁵⁵¹⁸</i> mutant embryos are able to recognize <i>E. coli</i> and can be seen to bind the fluorescently labelled bacteria (arrows) in a pattern indistinguishable from the wildtype. (D) Confocal images showing a series of optical slices taken through GFP expressing hemocytes. Images clearly show that the cells have internalised injected RFP labelled <i>E .coli</i> (arrows). (E) A projection of the slices shown in (D) highlight the two hemocytes (green) containing <i>E .coli</i> (red). (F) Z section taken through the region depicted by the dotted line on (E) clearly shows that the bacteria (red) are contained within the hemocyte (green). Arrows mark the cell extremities and correspond to the position of the arrows in (E). Scale bars represent 50 µm (A–C) and 10 µm (D and E).</p>", "links"=>[], "tags"=>["recognised", "engulfed", "embryonic"], "article_id"=>561231, "categories"=>["Immunology", "Cell Biology", "Developmental Biology"], "users"=>["Isabella Vlisidou", "Andrea J. Dowling", "Iwan R. Evans", "Nicholas Waterfield", "Richard H. ffrench-Constant", "Will Wood"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000518.g001", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Injected_E_coli_are_recognised_and_engulfed_by_embryonic_hemocytes_/561231", "title"=>"Injected <i>E. coli</i> are recognised and engulfed by embryonic hemocytes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-17 00:20:31"}
• {"files"=>["https://ndownloader.figshare.com/files/891218"], "description"=>"<p>(A) Stills from a movie showing dorsal closure in an embryo expressing GFP moesin in all tissues. Over a period of 2 hours opposing lateral epithelial sheets sweep dorsally and fuse in the midline to seal the dorsal side of the embryo. (B) Stills from a movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s007\" target=\"_blank\">Video S7</a>) showing high magnification detail of the boxed region in A show that opposing epithelial fronts zipper together at the canthi (arrowhead) to close the dorsal hole. Arrows point to a hemocyte as it migrates beneath the epithelium. (C) Stills from a timelapse movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s007\" target=\"_blank\">Video S7</a>) of an embryo injected with Mcf1 show that the presence of the toxin has no effect on dorsal closure which proceeds at the same speed as observed in wildtype embryos (compare with (A)). (D) High magnification stills from zipping canthi show that epithelial fronts fuse as normal in these areas. Note that no dynamic hemocytes can be seen in these stills nor in the accompanying movie due to the freezing effect of Mcf1 on these cells. Scale bars represent 50 µm (A and C) and 10 µm (B and D). Elapsed time is indicated in the upper right corner.</p>", "links"=>[], "tags"=>["dorsal"], "article_id"=>561678, "categories"=>["Immunology", "Cell Biology", "Developmental Biology"], "users"=>["Isabella Vlisidou", "Andrea J. Dowling", "Iwan R. Evans", "Nicholas Waterfield", "Richard H. ffrench-Constant", "Will Wood"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000518.g004", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mcf1_has_no_effect_on_dorsal_closure_/561678", "title"=>"Mcf1 has no effect on dorsal closure.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-17 00:27:58"}
• {"files"=>["https://ndownloader.figshare.com/files/890952"], "description"=>"<p>(A) Stills from a movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s002\" target=\"_blank\">Video S2</a>) of hemocytes expressing RFPmoesin following injection of GFP expressing <i>E. coli</i>. Hemocytes (asterisk) can be clearly seen actively migrating within the embryo and extending dynamic actin rich protrusions (arrow) as they bind and clear the injected <i>E. coli</i> (yellow). Insets show the movement of bacteria over the period of the movie as they are carried within the migrating hemocytes. (B) Stills from a movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s002\" target=\"_blank\">Video S2</a>) of RFP labelled hemocytes following injection of GFP labelled <i>Photorhabdus</i>. Hemocytes are able to recognise and bind the bacteria (yellow) but are completely frozen remaining in the same position for the duration of the movie (60 minutes). Cells still display actin rich protrusions (arrows) but these are static and bear no resemblance to the dynamic structures normally seen in hemocytes (compare to (A)). Insets show that the <i>Photorhabdus</i> bacteria themselves do not move throughout the movie as they are anchored to the static hemocytes. Scale bars represent 10 µm. Elapsed time is indicated in the upper right corner.</p>", "links"=>[], "tags"=>["injection", "causes", "freezing", "embryonic"], "article_id"=>561404, "categories"=>["Immunology", "Cell Biology", "Developmental Biology"], "users"=>["Isabella Vlisidou", "Andrea J. Dowling", "Iwan R. Evans", "Nicholas Waterfield", "Richard H. ffrench-Constant", "Will Wood"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000518.g002", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Photorhabdus_injection_causes_a_dramatic_freezing_of_embryonic_hemocytes_/561404", "title"=>"<i>Photorhabdus</i> injection causes a dramatic freezing of embryonic hemocytes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-17 00:23:24"}
• {"files"=>["https://ndownloader.figshare.com/files/441566", "https://ndownloader.figshare.com/files/441605", "https://ndownloader.figshare.com/files/441651", "https://ndownloader.figshare.com/files/441684", "https://ndownloader.figshare.com/files/441718", "https://ndownloader.figshare.com/files/441747", "https://ndownloader.figshare.com/files/441786", "https://ndownloader.figshare.com/files/441821", "https://ndownloader.figshare.com/files/441859", "https://ndownloader.figshare.com/files/441895"], "description"=>"<div><p><em>Drosophila</em> embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (<em>Photorhabdus asymbiotica</em>) and non-pathogenic (<em>Escherichia coli</em>) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic <em>E. coli</em> in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen <em>Photorhabdus</em> leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant <em>E. coli</em> expressing the <em>mcf1</em> gene. Mcf1 mediated hemocyte freezing is <em>shibire</em> dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how <em>Drosophila</em> embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.</p> </div>", "links"=>[], "tags"=>["embryos", "systems", "monitoring", "bacterial"], "article_id"=>147039, "categories"=>["Immunology", "Cell Biology", "Developmental Biology"], "users"=>["Isabella Vlisidou", "Andrea J. Dowling", "Iwan R. Evans", "Nicholas Waterfield", "Richard H. ffrench-Constant", "Will Wood"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1000518.s001", "https://dx.doi.org/10.1371/journal.ppat.1000518.s002", "https://dx.doi.org/10.1371/journal.ppat.1000518.s003", "https://dx.doi.org/10.1371/journal.ppat.1000518.s004", "https://dx.doi.org/10.1371/journal.ppat.1000518.s005", "https://dx.doi.org/10.1371/journal.ppat.1000518.s006", "https://dx.doi.org/10.1371/journal.ppat.1000518.s007", "https://dx.doi.org/10.1371/journal.ppat.1000518.s008", "https://dx.doi.org/10.1371/journal.ppat.1000518.s009", "https://dx.doi.org/10.1371/journal.ppat.1000518.s010"], "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Drosophila_Embryos_as_Model_Systems_for_Monitoring_Bacterial_Infection_in_Real_Time/147039", "title"=>"<em>Drosophila</em> Embryos as Model Systems for Monitoring Bacterial Infection in Real Time", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2009-07-17 01:57:19"}
• {"files"=>["https://ndownloader.figshare.com/files/891096"], "description"=>"<p>(A) Stills from a movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s003\" target=\"_blank\">Video S3</a>) of hemocytes following injection of <i>E. coli</i> expressing Mcf1. The images clearly show that whilst the hemocytes can recognise the bacteria and bind them the cells are apparently frozen and fail to move throughout the whole 60 minutes of the movie. Insets show that the position of the bacteria does not change over the course of the movie. (B) Stills from a movie (see <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000518#ppat.1000518.s004\" target=\"_blank\">Video S4</a>) of GFPmoesin expressing hemocytes following injection of purified Mcf1 show an identical effect to that seen in (A). Hemocytes are unable to move, have static actin rich protrusions (arrows) and over time accumulate GFP positive puncta in the cytoplasm (arrowheads). (C) Graph showing percentage mortality of embryos following Mcf1 injection. 75% of embryos fail to hatch following Mcf1 injection as opposed to 26% of those injected with heat inactivated BSA. (D) Embryos containing GFP expressing hemocytes (arrowheads) 12 hours after injection with Mcf1 (below) show a dramatic reduction in hemocyte number when compared with control embryos (above). (E) Hemocytes expressing UAS-Bax to induce apoptosis are morphologically distinct from hemocytes exposed to Mcf1 (compare with B) appearing circular and lacking any protrusive structures (arrow). (F) Confocal image of hemocytes within a dead embryo 12 hours after Mcf1 injection. Hemocytes appear identical to apoptotic cells expressing Bax (compare with E). Scale bars represent 10 µm (A, B, E and F) and 100 µm (D). Elapsed time is indicated in the upper right corner in A and B.</p>", "links"=>[], "tags"=>["expressing", "mcf1", "recognised", "hemocytes", "induce", "freezing"], "article_id"=>561545, "categories"=>["Immunology", "Cell Biology", "Developmental Biology"], "users"=>["Isabella Vlisidou", "Andrea J. Dowling", "Iwan R. Evans", "Nicholas Waterfield", "Richard H. ffrench-Constant", "Will Wood"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000518.g003", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_E_coli_expressing_Mcf1_are_recognised_by_hemocytes_and_induce_a_freezing_phenotype_/561545", "title"=>"<i>E. coli</i> expressing Mcf1 are recognised by hemocytes and induce a freezing phenotype.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-17 00:25:45"}

PMC Usage Stats | Further Information

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Relative Metric

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F1000Prime | Further Information

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