Identification of genetic modifiers of CagA-induced epithelial disruption in Drosophila

Abstract

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 (EGFR), 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.

Keywords: CagA; Drosophila; Helicobacter pylori; coracle; crumbs; epithelia; genetic modifier.

Figure 1

(A) Crossing scheme for the Moc deficiency screen. Flies containing the genetic deficiency were compared to those containing a visual marker such as CyO. Flies expressing CagA in a wild-type, (B) or egfr^−/+ background, (C) were imaged by ESEM. (D) Chromosomal map of the genetic deficiency screen. The result from each deficiency (darker colors) is indicated along with the inferred functionality of each genetic region (lighter colors), where deficiencies that caused no change override those that cause enhancement or suppression.

Figure 2

(A–F) Representative ESEM images for each class of disruption by CagA. The scoring rubric is described in Materials and Methods. (G) The mean ESEM-based eye disruption for CagA, known interactors and Moc genes of different functional classes. Error bars represent standard error.

Figure 3

(A) Pattern of Dlg staining in Z-stacks of larval retinal imaginal discs. CagA-expressing eye discs are compared to those expressing the GMR-Gal4 driver alone. Shaded areas represent standard error. The arrow indicates the point at 4.8 microns below the peak intensity where the distribution was evaluated. (B) Quantification of larval retinal epithelial morphology for par1 and cora mutants, and their interactions with CagA.

Figure 4

cora reduction suppresses CagA-induced epithelial disorganization but not CagA protein localization to septate junctions. (A–C) Model for the basal displacement of Dlg. Panel A represents the wild-type distribution of Dlg (represented as red structures on the lateral membranes of the epithelial cells). Panel B represents basally expanded Dlg expression due to expansion within individual cells. Panel C shows how epithelial disruption can cause basal mispositioning of Dlg expression by positioning cells deeper within the epithelium. (D) Control larval retinal epithelium (GMR-Gal4) stained with Dlg (red) and E-cad (green). YZ and XZ orthogonal planes are shown on the side and top, respectively, in D and E. Scale bar is 30 microns for all panels. (E) CagA-expressing larval retinal epithelium (GMR-Gal4; UAS-CagA) also stained with Dlg (red) and E-cad (green). Arrowhead in the upper orthogonal section shows basally mispositioned Dlg staining. Arrow indicates Dlg staining that is deep within the epithelium due to irregularities in the epithelial sheet. (F) cora^+/− larval retinal epithelium expressing CagA (GMR-Gal4; UAS-CagA) showing CagA localization as labeled with anti-HA. Apical HA puncta are present. (G) A larval retinal disc expressing CagA (GMR-Gal4; UAS-CagA) labeled with HA antibody.

Figure 5

(A) Interactions of several epithelial polarity determinants with CagA, using the Dlg distribution assay described in Figure 3. Error bars represent standard error. Green asterisks represent mutants that significantly suppress CagA-induced epithelial disruption; red asterisks represent mutants that significantly enhance epithelial disruption (p < 0.05). (B) Model for the interactions of CagA with epithelial polarity determinants Cora and Crb.