Publications by authors named "Annabel Guichard"

14 Publications

  • Page 1 of 1

A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways.

iScience 2020 Feb 25;23(2):100865. Epub 2020 Jan 25.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0335, USA; Tata Institute for Genetics and Society-UCSD, La Jolla, CA 92093-0335, USA. Electronic address:

Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.
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http://dx.doi.org/10.1016/j.isci.2020.100865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011083PMC
February 2020

Efficient allelic-drive in Drosophila.

Nat Commun 2019 04 9;10(1):1640. Epub 2019 Apr 9.

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0335, USA.

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.
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http://dx.doi.org/10.1038/s41467-019-09694-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456580PMC
April 2019

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

PLoS Pathog 2017 Sep 25;13(9):e1006603. Epub 2017 Sep 25.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, United States of America.

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.
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http://dx.doi.org/10.1371/journal.ppat.1006603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612732PMC
September 2017

Influenza NS1 directly modulates Hedgehog signaling during infection.

PLoS Pathog 2017 Aug 24;13(8):e1006588. Epub 2017 Aug 24.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

The multifunctional NS1 protein of influenza A viruses suppresses host cellular defense mechanisms and subverts other cellular functions. We report here on a new role for NS1 in modifying cell-cell signaling via the Hedgehog (Hh) pathway. Genetic epistasis experiments and FRET-FLIM assays in Drosophila suggest that NS1 interacts directly with the transcriptional mediator, Ci/Gli1. We further confirmed that Hh target genes are activated cell-autonomously in transfected human lung epithelial cells expressing NS1, and in infected mouse lungs. We identified a point mutation in NS1, A122V, that modulates this activity in a context-dependent fashion. When the A122V mutation was incorporated into a mouse-adapted influenza A virus, it cell-autonomously enhanced expression of some Hh targets in the mouse lung, including IL6, and hastened lethality. These results indicate that, in addition to its multiple intracellular functions, NS1 also modifies a highly conserved signaling pathway, at least in part via cell autonomous activities. We discuss how this new Hh modulating function of NS1 may influence host lethality, possibly through controlling cytokine production, and how these new insights provide potential strategies for combating infection.
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http://dx.doi.org/10.1371/journal.ppat.1006588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587344PMC
August 2017

RAB11-mediated trafficking in host-pathogen interactions.

Nat Rev Microbiol 2014 Sep;12(9):624-34

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

Many bacterial and viral pathogens block or subvert host cellular processes to promote successful infection. One host protein that is targeted by invading pathogens is the small GTPase RAB11, which functions in vesicular trafficking. RAB11 functions in conjunction with a protein complex known as the exocyst to mediate terminal steps in cargo transport via the recycling endosome to cell-cell junctions, phagosomes and cellular protrusions. These processes contribute to host innate immunity by promoting epithelial and endothelial barrier integrity, sensing and immobilizing pathogens and repairing pathogen-induced cellular damage. In this Review, we discuss the various mechanisms that pathogens have evolved to disrupt or subvert RAB11-dependent pathways as part of their infection strategy.
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http://dx.doi.org/10.1038/nrmicro3325DOI Listing
September 2014

Cholera toxin disrupts barrier function by inhibiting exocyst-mediated trafficking of host proteins to intestinal cell junctions.

Cell Host Microbe 2013 Sep;14(3):294-305

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

Cholera toxin (CT), a virulence factor elaborated by Vibrio cholerae, is sufficient to induce the severe diarrhea characteristic of cholera. The enzymatic moiety of CT (CtxA) increases cAMP synthesis in intestinal epithelial cells, leading to chloride ion (Cl(-)) efflux through the CFTR Cl(-) channel. To preserve electroneutrality and osmotic balance, sodium ions and water also flow into the intestinal lumen via a paracellular route. We find that CtxA-driven cAMP increase also inhibits Rab11/exocyst-mediated trafficking of host proteins including E-cadherin and Notch signaling components to cell-cell junctions in Drosophila, human intestinal epithelial cells, and ligated mouse ileal loops, thereby disrupting barrier function. Additionally, CtxA induces junctional damage, weight loss, and dye leakage in the Drosophila gut, contributing to lethality from live V. cholerae infection, all of which can be rescued by Rab11 overexpression. These barrier-disrupting effects of CtxA may act in parallel with Cl(-) secretion to drive the pathophysiology of cholera.
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http://dx.doi.org/10.1016/j.chom.2013.08.001DOI Listing
September 2013

Deconstructing host-pathogen interactions in Drosophila.

Dis Model Mech 2012 Jan 6;5(1):48-61. Epub 2011 Oct 6.

University of California, San Diego, La Jolla, CA 92039, USA.

Many of the cellular mechanisms underlying host responses to pathogens have been well conserved during evolution. As a result, Drosophila can be used to deconstruct many of the key events in host-pathogen interactions by using a wealth of well-developed molecular and genetic tools. In this review, we aim to emphasize the great leverage provided by the suite of genomic and classical genetic approaches available in flies for decoding details of host-pathogen interactions; these findings can then be applied to studies in higher organisms. We first briefly summarize the general strategies by which Drosophila resists and responds to pathogens. We then focus on how recently developed genome-wide RNA interference (RNAi) screens conducted in cells and flies, combined with classical genetic methods, have provided molecular insight into host-pathogen interactions, covering examples of bacteria, fungi and viruses. Finally, we discuss novel strategies for how flies can be used as a tool to examine how specific isolated virulence factors act on an intact host.
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http://dx.doi.org/10.1242/dmm.000406DOI Listing
January 2012

New insights into the biological effects of anthrax toxins: linking cellular to organismal responses.

Microbes Infect 2012 Feb 8;14(2):97-118. Epub 2011 Sep 8.

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA.

The anthrax toxins lethal toxin (LT) and edema toxin (ET) are essential virulence factors produced by Bacillus anthracis. These toxins act during two distinct phases of anthrax infection. During the first, prodromal phase, which is often asymptomatic, anthrax toxins act on cells of the immune system to help the pathogen establish infection. Then, during the rapidly progressing (or fulminant) stage of the disease bacteria disseminate via a hematological route to various target tissues and organs, which are typically highly vascularized. As bacteria proliferate in the bloodstream, LT and ET begin to accumulate rapidly reaching a critical threshold level that will cause death even when the bacterial proliferation is curtailed by antibiotics. During this final phase of infection the toxins cause an increase in vascular permeability and a decrease in function of target organs including the heart, spleen, kidney, adrenal gland, and brain. In this review, we examine the various biological effects of anthrax toxins, focusing on the fulminant stage of the disease and on mechanisms by which the two toxins may collaborate to cause cardiovascular collapse. We discuss normal mechanisms involved in maintaining vascular integrity and based on recent studies indicating that LT and ET cooperatively inhibit membrane trafficking to cell-cell junctions we explore several potential mechanisms by which the toxins may achieve their lethal effects. We also summarize the effects of other potential virulence factors secreted by B. anthracis and consider the role of toxic factors in the evolutionarily recent emergence of this devastating disease.
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http://dx.doi.org/10.1016/j.micinf.2011.08.016DOI Listing
February 2012

Anthrax toxins cooperatively inhibit endocytic recycling by the Rab11/Sec15 exocyst.

Nature 2010 Oct;467(7317):854-8

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0349, USA.

Bacillus anthracis is the causative agent of anthrax in humans and other mammals. In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and oedema factor (EF), leading to widespread vascular leakage and shock. Whereas host targets of LF (mitogen-activated protein-kinase kinases) and EF (cAMP-dependent processes) have been implicated in the initial phase of anthrax, less is understood about toxin action during the final stage of infection. Here we use Drosophila melanogaster to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF. EF reduces levels of apically localized Rab11 and indirectly blocks vesicle formation by its binding partner and effector Sec15 (Sec15-GFP), whereas LF acts more directly to reduce Sec15-GFP vesicles. Convergent effects of EF and LF on Rab11/Sec15 inhibit expression of and signalling by the Notch ligand Delta and reduce DE-cadherin levels at adherens junctions. In human endothelial cells, the two toxins act in a conserved fashion to block formation of Sec15 vesicles, inhibit Notch signalling, and reduce cadherin expression at adherens junctions. This coordinated disruption of the Rab11/Sec15 exocyst by anthrax toxins may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.
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http://dx.doi.org/10.1038/nature09446DOI Listing
October 2010

Dynein and Star interact in EGFR signaling and ligand trafficking.

J Cell Sci 2008 Aug 24;121(Pt 16):2643-51. Epub 2008 Jul 24.

University of Minnesota, Department of Genetics, Cell Biology and Development, Minneapolis, MN 55455, USA.

Intracellular transport and processing of ligands is critical to the activation of signal transduction pathways that guide development. Star is an essential gene in Drosophila that has been implicated in the trafficking of ligands for epidermal growth factor (EGF) receptor signaling. The role of cytoplasmic motors in the endocytic and secretory pathways is well known, but the specific requirement of motors in EGF receptor transport has not been investigated. We identified Star in a screen designed to recover second-site modifiers of the dominant rough eye phenotype of the Glued mutation Gl(1). The Glued (Gl) locus encodes the p150 subunit of the dynactin complex, an activator of cytoplasmic dynein-driven motility. We show that alleles of Gl and dynein genetically interact with both Star and EGFR alleles. Similarly to mutations in Star, the Gl(1) mutation is capable of modifying the phenotypes of the EGFR mutation Ellipse. These genetic interactions suggest a model in which Star, dynactin and dynein cooperate in the trafficking of EGF ligands. In support of this model, overexpression of the cleaved, active Spitz ligand can partially bypass defective trafficking and suppress the genetic interactions. Our direct observations of live S2 cells show that export of Spitz-GFP from the endoplasmic reticulum, as well as the trafficking of Spitz-GFP vesicles, depends on both Star and dynein.
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http://dx.doi.org/10.1242/jcs.027144DOI Listing
August 2008

Anthrax lethal factor and edema factor act on conserved targets in Drosophila.

Proc Natl Acad Sci U S A 2006 Feb 2;103(9):3244-9. Epub 2006 Feb 2.

Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, 92093-0349, USA.

Many bacterial toxins act on conserved components of essential host-signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila melanogaster can be used for analyzing the mechanism of toxin action. In this study, we characterize the activities of two anthrax virulence factors, lethal factor (LF) and edema factor, in transgenic Drosophila. LF is a zinc metalloprotease that cleaves and inactivates most human mitogen-activated protein kinase (MAPK) kinases (MAPKKs). We found that LF similarly cleaves the Drosophila MAPK kinases Hemipterous (Hep) and Licorne in vitro. Consistent with these observations, expression of LF in Drosophila inhibited the Hep/c-Jun N-terminal kinase pathway during embryonic dorsal closure and the related process of adult thoracic closure. Epistasis experiments confirmed that LF acts at the level of Hep. We also found that LF inhibits Ras/MAPK signaling during wing development and that LF acts upstream of MAPK and downstream of Raf, consistent with LF acting at the level of Dsor. In addition, we found that edema factor, a potent adenylate cyclase, inhibits the hh pathway during wing development, consistent with the known role of cAMP-dependent PKA in suppressing the Hedgehog response. These results demonstrate that anthrax toxins function in Drosophila as they do in mammalian cells and open the way to using Drosophila as a multicellular host system for studying the in vivo function of diverse toxins and virulence factors.
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http://dx.doi.org/10.1073/pnas.0510748103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1413899PMC
February 2006

Characterization of a human rhomboid homolog, p100hRho/RHBDF1, which interacts with TGF-alpha family ligands.

Dev Dyn 2005 Aug;233(4):1315-31

Department of Cell and Tissue Biology, Program in Cell Biology, University of California at San Francisco, San Francisco, California 94143-0512, USA.

The activity of the TGF-alpha-like ligand Spitz in Drosophila depends on Rhomboid, a seven-transmembrane spanning protein that resides in the Golgi and acts as a serine protease to cleave Spitz, thereby releasing the soluble ligand. Several rhomboids in Drosophila have been implicated in the processing of TGF-alpha-like ligands, and consequent EGF receptor activation. The larger number of TGF-alpha-like ligands in vertebrates raises the possibility that they too might be subject to regulation by rhomboid-like proteins. We present the cDNA cloning and polypeptide sequence of an atypically long human rhomboid, which, based on the absence of critical residues for serine protease activity, is not predicted to act as a serine protease. We examined its tissue distribution, in comparison with TGF-alpha and the TGF-alpha-related protein HB-EGF, and the EGF/TGF-alpha receptor, in mouse embryo. This rhomboid, named p100(hRho) or RHBDF1, is a seven-transmembrane protein with a long N-terminal cytoplasmic extension that comprises half of the polypeptide sequence, and is found in the endoplasmic reticulum and Golgi, but not on the cell surface. It is expressed as two forms with different lengths, forms dimers and interacts with TGF-alpha ligands through a luminal interaction with the EGF core ectodomain. Finally, we evaluated the function of p100(hRho)/RHBDF1 in Drosophila, demonstrating that the short, but not the full-length form has functional activity. The characterization of this protein extends our understanding of the rhomboid family of regulatory proteins.
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http://dx.doi.org/10.1002/dvdy.20450DOI Listing
August 2005

Activation of the knirps locus links patterning to morphogenesis of the second wing vein in Drosophila.

Development 2003 Jan;130(2):235-48

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA.

The adjacent knirps (kni) and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expressing high levels of the related spalt genes. Here, we identify a 1.4 kb cis-acting enhancer element from the kni locus that faithfully directs gene expression in the L2 primordium. We find that three independent ri alleles have alterations mapping within the L2-enhancer element and show that two of these observed lesions eliminate the ability of the enhancer element to direct gene expression in the L2 primordium. The L2 enhancer can be subdivided into distinct activation and repression domains. The activation domain mediates the combined action of the general wing activator Scalloped and a putative locally provided factor, the activity of which is abrogated by a single nucleotide alteration in the ri(53j) mutant. We also find that misexpression of genes in L2 that are normally expressed in veins other than L2 results in abnormal L2 development. These experiments provide a mechanistic basis for understanding how kni and knrl link AP patterning to morphogenesis of the L2 vein by orchestrating the expression of a selective subset of vein-promoting genes in the L2 primordium.
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http://dx.doi.org/10.1242/dev.00207DOI Listing
January 2003

A screen for dominant mutations applied to components in the Drosophila EGF-R pathway.

Proc Natl Acad Sci U S A 2002 Mar;99(6):3752-7

Section of Cell and Developmental Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA.

The Drosophila epidermal growth factor receptor (EGF-R) controls many critical cell fate choices throughout development. Several proteins collaborate to promote localized EGF-R activation, such as Star and Rhomboid (Rho), which act sequentially to ensure the maturation and processing of inactive membrane-bound EGF ligands. To gain insights into the mechanisms underlying Rho and Star function, we developed a mutagenesis scheme to isolate novel overexpression activity (NOVA) alleles. In the case of rho, we isolated a dominant neomorphic allele, which interferes with Notch signaling, as well as a dominant-negative allele, which produces RNA interference-like flip-back transcripts that reduce endogenous rho expression. We also obtained dominant-negative and neomorphic Star mutations, which have phenotypes similar to those of rho NOVA alleles, as well as dominant-negative Egf-r alleles. The isolation of dominant alleles in several different genes suggests that NOVA mutagenesis should be widely applicable and emerge as an effective tool for generating dominant mutations in genes of unknown function.
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http://dx.doi.org/10.1073/pnas.052028699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122596PMC
March 2002
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