Helicobacter pylori CagA Disrupts Epithelial Patterning by Activating Myosin Light Chain

Helicobacter pylori infection is a leading cause of ulcers and gastric cancer. We show that expression of the H. pylori virulence factor CagA in a model Drosophila melanogaster epithelium induces morphological disruptions including ectopic furrowing. We find that CagA alters the distribution and increases the levels of activated myosin regulatory light chain (MLC), a key regulator of epithelial integrity. Reducing MLC activity suppresses CagA-induced disruptions. A CagA mutant lacking EPIYA motifs (CagAEPISA) induces less epithelial disruption and is not targeted to apical foci like wild-type CagA. In a cell culture model in which CagAEPISA and CagA have equivalent subcellular localization, CagAEPISA is equally potent in activating MLC. Therefore, in our transgenic system, CagA is targeted by EPIYA motifs to a specific apical region of the epithelium where it efficiently activates MLC to disrupt epithelial integrity

MLC regulation in CagA-expressing eye epithelia.

<p>(A) Deep confocal section of a control eye epithelium (GMR-Gal4) expressing MLC-GFP. Cross section reveals enrichment of MLC-GFP along the apical surface of the epithelium beginning at the MF. Scale bar is 50 uM. (B) Deep confocal section of a GMR-Gal4; UAS-CagA expressing eye epithelium. Confocal plane goes through an ectopic furrow revealing the infolded apical surfaces of the epithelium expressing enriched MLC-GFP. Cross section reveals apical MLC-GFP expression and enrichment of MLC-GFP in the ectopic furrow. (C) Flattened confocal stack of a control (GMR-Gal4) eye epithelium stained with anti-MLCpp (green) and phalloidin (red) to mark the MF. Cross section reveals MLCpp expression within the MF. Only very weak expression of MLCpp is present posterior to the furrow. Scale bar is 100 uM. (D) Flattened confocal stack of a CagA (GMR-Gal4; UAS-CagA) expressing eye epithelium displaying a complex pattern of MLCpp expression posterior to the MF. Optical cross section reveals MLCpp expression within an ectopic furrow.</p

CagA induces rapid epithelial disruption.

<p>(A) Schematic of third instar larval eye disc development, showing the anterior advance of the morphogenetic furrow (MF). As the MF advances, undifferentiated cells (red cells on left) differentiate into photoreceptors (depicted as purple dots). (B) Cross section of a third instar larval epithelium labeled with F-actin (green) and ElaV (red) to mark photoreceptor nuclei. All images are oriented with anterior to the left. The MF advances in the direction of the arrow as development proceeds. The asterisk marks a punctum of actin apical to the photoreceptors. (C) A 3D reconstruction of a third instar larval eye disc expressing CagA-HA, as labeled by anti-HA. (D) XY confocal plane of a control eye epithelium expressing GMR-Gal4 alone. Photoreceptors (red) are spatially separated. The MF is positioned at the far left in D-F. Image below shows an optical cross section of a GMR-Gal4 eye epithelium showing planar arrangement of photoreceptor clusters (red) that each contact an apical actin punctum (green). (E) GMR-Gal4; UAS-CagA expressing eye epithelium displaying improper separation of actin foci into what appear at this resolution to be long bands of continuous actin. Lower panel shows a cross section of a GMR-Gal4;UAS-CagA expressing eye epithelium showing an ectopic furrow displacing photoreceptor nuclei basally. (F) GMR-Gal4; UAS-CagA*2 expressing eye disc displaying a deep ectopic furrow. Lower panel shows a cross section of a GMR-Gal4; UAS-CagA*2 expressing eye epithelium with photoreceptor nuclei displaced basally. (G) A superficial confocal plane of a GMR-Gal4 control eye disc showing PH-GFP expression surrounding ommatidia. (H) A deep confocal section showing the underlying photoreceptor cells and the absence of ElaV positive cells within the deep layers of the tissue. (I) Superficial confocal section of a GMR-Gal4; UAS-CagA eye epithelium (J) A deep section of a GMR-Gal4; UAS-CagA eye epithelium showing ElaV –positive cells within the deep region of the epithelium and interspersed with PH-GFP expressing cells. (K) An optical cross section through a GMR-Gal4 control eye epithelium showing the arrangement of PH-GFP and ElaV expressing cells. (L) An optical cross section through a GMR-Gal4; UAS-CagA expressing eye epithelium showing the apicobasal mispositioning of ElaV and PH-GFP expressing cells. Scale bars in D and G are 50 microns.</p

CagA interactions with MLC and the MLC activator, Rok.

<p>(A) Eye epithelium expressing an inactivating MLC mutation (<i>sqhA21</i>) displaying normal morphology. ElaV (red) labels photoreceptors and phalloidin (green) labels F-actin. Scale bar is 50 microns. (B) GMR-Gal4; UAS-CagA expressing eye epithelium displaying ectopic furrowing. (C) GMR-Gal4;UAS-CagA expressing eye epithelium co-expressing a single copy of <i>sqhA21</i> displaying very mild ectopic furrowing. (D) GMR-Gal4; UAS-RokCAT epithelium with ectopic furrows. (E) GMR-Gal4; UAS-CagA and UAS-RokCAT displaying severe ectopic furrowing. (F) GMR-Gal4; UAS-Rho^CA displaying moderate ectopic furrowing. (G) Quantification: The area of deep photoreceptors was determined by first inverting a 3D reconstruction of each eye epithelium, and determining the area of ElaV expression in Image J. This value was divided by the total area of the eye epithelium, thus providing a metric for morphological disruption. * indicates statistical significance. P value for CagA vs CagA; sqhA21^(-/-) is less than 0.0001, and for CagA vs CagA; RokCAT p value is 0.0005.</p

CagA induces Rho-dependent MLC-GFP relocalization in S2 cells.

<p>(A) Control S2 cells plated on ConA expressing MLC-GFP predominantly in the periphery of cell. Cell labeled i is representative of the intermediate phenotype. Scale bar: 25 microns. (B) A CagA transfected S2 cell expressing MLC-GFP in a central ring surrounding the nucleus. Cell labeled r is representative of the central ring phenotype. (C) High magnifcation optical cross section of a control MLC-GFP expressing S2 cell showing MLC-GFP expression in close association with the substrate (labeled with a transparent line). (D) High magnification optical cross section of a control MLC-GFP expressing S2 cell showing MLC-GFP expression above the substrate. (E) Graph showing the relative distribution of cell types (diffuse, central ring, or intermediate) in each treatment. (F) A Rho RNAi treated S2 cell showing diffuse MLC-GFP expression. Cell below d is representative of the diffuse phenotype. (G) A CagA transfected cell treated with Rho RNAi showing diffuse MLC-GFP expression. (H) A Rok RNAi treated S2 cell showing diffuse MLC-GFP expression (I) A CagA transfected cell treated with Rok RNAi showing diffuse MLC-GFP expression. (J) A Rok inhibitior (Y-27632)-treated cell showing diffuse MLC-GFP expression. (K) A Rok inhibitor treated cell showing diffuse MLC-GFP expression.</p

CagA and CagA^EPISA expression in MLC-GFP-expressing S2 cells.

<p>(A) CagA expressing S2 cell marked with HA antibody (red). In the transfected cell, MLC-GFP (green) is expressed in a “central ring.” (B) A CagA^EPISA expressing cell showing the central ring MLC-GFP pattern. Scale bar: 5 microns.</p

CagA localization is directed by EPIYA motifs.

<p>(A) GMR-Gal4; UAS-CagA expressing eye epithelium labeled with anti-HA to reveal the pattern of CagA expression. Scale bar for A, B, D, E: 50 microns. (B) YZ optical section of (A) showing HA expression in apical punctae. (C) A high magnification view of (A) showing an ommatidium with HA expression concentrated at the apical foci. (D) GMR-Gal4; UAS-CagA^EPISA expressing eye epithelium. (E) YZ optical section of (D) showing diffuse, cytoplasmic expression in individual ommatidial cells. (F) A high magnification view of (E) showing HA expression throughout the cell. (G) <i>slbo</i>-Gal4; UAS-GFP, UAS-CagA expressing follicular epithelial cell. (H) Cortical enrichment of HA expression in follicular epithelial cells. (I) <i>slbo</i>-Gal4: UAS-GFP, UAS-CagA^EPISA expressing follicular epithelial cells. (J) Cortical enrichment of HA expression in CagA^EPISA expressing follicular epithelial cells. (K) GMR-Gal4; UAS-CagA^EPISA expressing eye epithelium showing high HA expression in a subset of photoreceptors. (L) GMR-Gal4; UAS-PH-GFP expressing eye epithelium showing GFP expression surrounding the ommatidia but not in photoreceptors. Scale bar in K and L: 10 microns.</p

Characterization of genetic manipulations of the cytoskeleton in the eye disc.

<p>Characterization of genetic manipulations of the cytoskeleton in the eye disc.</p

Identification of Genetic Modifiers of CagA-Induced Epithelial Disruption in Drosophila

Helicobacter pylori strains containing the CagA protein are associated with high risk of gastric diseases including atrophic gastritis, peptic ulcers, and gastric cancer. CagA is injected into host cells via a Type IV secretion system where it activates growth factor-like signaling, disrupts cell-cell junctions, and perturbs host cell polarity. Using a transgenic Drosophila model, we have shown that CagA expression disrupts the morphogenesis of epithelial tissues such as the adult eye. Here we describe a genetic screen to identify modifiers of CagA-induced eye defects. We determined that reducing the copy number of genes encoding components of signaling pathways known to be targeted by CagA, such as the epidermal growth factor receptor, modified the CagA-induced eye phenotypes. In our screen of just over half the Drosophila genome, we discovered 12 genes that either suppressed or enhanced CagA’s disruption of the eye epithelium. Included in this list are genes involved in epithelial integrity, intracellular trafficking, and signal transduction. We investigated the mechanism of one suppressor, encoding the epithelial polarity determinant and junction protein Coracle, which is homologous to the mammalian Protein 4.1. We found that loss of a single copy of coracle improved the organization and integrity of larval retinal epithelia expressing CagA, but did not alter CagA’s localization to cell junctions. Loss of a single copy of the coracle antagonist crumbs enhanced CagA-associated disruption of the larval retinal epithelium, whereas overexpression of crumbs suppressed this phenotype. Collectively, these results point to new cellular pathways whose disruption by CagA are likely to contribute to H. pylori-associated disease pathology