Abstract
Cortactin is an actin-binding protein and actin-nucleation promoting factor regulating cytoskeletal rearrangements in eukaryotes. Helicobacter pylori is a gastric pathogen that exploits cortactin to its own benefit. During infection of gastric epithelial cells, H. pylori hijacks multiple cellular signaling pathways, leading to the disruption of key cell functions. Two bacterial virulence factors play important roles in this scenario, the vacuolating cytotoxin VacA and the translocated effector protein CagA of the cag type IV secretion system (T4SS). Specifically, by overruling the phosphorylation status of cortactin, H. pylori alternates the activity of molecular interaction partners of this important protein, thereby manipulating the performance of cytoskeletal rearrangements, endosomal trafficking and cell movement. Based on shRNA knockdown and other studies, it was previously reported that VacA utilizes cortactin for its cellular uptake, intracellular travel and induction of apoptosis by a mitochondria-dependent mechanism, while CagA induces cell scattering, motility and elongation. To investigate the role of cortactin in these phenotypes in more detail, we produced a complete knockout mutant of cortactin in the gastric adenocarcinoma cell line AGS by CRISPR-Cas9. These cells were infected with H. pylori wild-type or various isogenic mutant strains. Unexpectedly, cortactin deficiency did not prevent the uptake and formation of VacA-dependent vacuoles, nor the induction of apoptosis by internalized VacA, while the induction of T4SS- and CagA-dependent AGS cell movement and elongation were strongly reduced. Thus, we provide evidence that cortactin is required for the function of internalized CagA, but not VacA.
Highlights
Helicobacter pylori represents one of the most successful pathogenic bacteria in humans; about half of the world’s population is infected by this microbe [1]
CagA is encoded by the so-called cag pathogenicity island, along with a multitude of other proteins (CagY, CagL, CagE, etc.), which form a type IV secretion system (T4SS) to deliver CagA in the host cell [15,16,17,18]
We aimed to take a closer look at the role of cortactin during infection with H. pylori, with specific interest regarding the functions of CagA and VacA
Summary
Helicobacter pylori represents one of the most successful pathogenic bacteria in humans; about half of the world’s population is infected by this microbe [1]. After injection by the T4SS, CagA becomes tyrosine-phosphorylated by members of the Src and Abl family of kinases [20,21,22] and can interact with a plethora of different host cell proteins, both in a phosphorylation-dependent and -independent manner to manipulate host cell signaling [23,24]. Deletion of the cttn gene in AGS cells disrupted H. pylori-induced signaling, in particular, by diminishing the activation levels of FAK, Src and Abl tyrosine kinases. We show that cortactin deficiency did not negatively affect the uptake or intracellular function of VacA during infection with H. pylori, or by addition of the purified protein, but did diminish the expression of the CagA-dependent elongation phenotype in AGS cells. The VacA-dependent apoptosis and vacuolization rates during H. pylori infection or treatments with purified VacA were found to be at similar rates in both the wt and cortactin-deficient AGS cells. The presented data shows that cortactin is rather involved in CagA-mediated AGS cell scattering signaling, while its role in VacA-mediated vacuolization and apoptosis seems to be dispensable
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