Science Bite (3 minute oral presentation with PPT in live session and poster) - Students and ECRs only 14th Lorne Infection and Immunity 2024

Just the Tipα of the iceberg: nuclear targeting by an Helicobacter pylori extracellular vesicle-associated toxin. (#51)

Jack Emery 1 2 , Variya Nemidkanam 2 3 , Nina Colon 2 , Dongmei Tong 2 , Caroline Skene 2 , Laurent Terradot 4 , Richard Ferrero 1 2 5
  1. Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia
  2. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia
  3. Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
  4. Molecular Microbiology and Structural Biochemistry, Institut de Biologie et Chimie des Protéines, Université de Lyon, Lyon, Cedex, France
  5. Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, VIC, Australia

Pathogenic bacteria produce a family of toxins, ‘nucleomodulins’, that target the nuclear compartment of host cells to cause epigenetic changes or modulate nuclear functions. While studies have examined how nucleomodulins alter nuclear processes, their intracellular trafficking is less defined. Recombinant forms of the Helicobacter pylori tumour necrosis factor-a-inducing protein (Tipa) were reported to translocate to the host cell nucleus, leading to tumour necrosis factor gene expression and carcinogenic effects. Furthermore, it was reported recombinant Tipa (rTipa) bound to surface expressed nucleolin as a receptor for internalisation in gastric cancer cells. Although Tipa is secreted by H. pylori, we observed in proteomic studies that it is also packaged within bacterial Extracellular Vesicles (EVs); membrane ‘blebs’ that are highly efficient at entering and subverting host cell functions. We hypothesised that H. pylori utilises EVs as a system to deliver Tipa to the nucleus, promoting carcinogenesis. To address this hypothesis, we characterised the secretion of Tipa by immunoblotting and observed that the majority of secreted Tipa was packaged within EVs. Next, we screened a collection of gastric epithelial cells for surface expression of nucleolin and identified MKN28 cells as expressing more nucleolin than other cell lines. Despite this, we observed by confocal microscopy and co-immunoprecipitation that rTipa localised to the perinuclear region of MKN-28 cells without binding nucleolin for internalisation. To determine the intracellular trafficking of Tipa, we cultured AGS gastric epithelial cells with H. pylori EVs or rTipa and observed by immunoblotting that Tipa associated with the nuclear compartment within 4 hours post-treatment. Importantly, recombinant and EV-associated Tipa trafficked through the golgi apparatus and endoplasmic reticulum to deliver Tipa to the nuclear compartment as evidenced by confocal microscopy. Taken together, we propose Tipa is an EV-associated nucleomodulin which targets the nucleus to promote gastric carcinogenesis.