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

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 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