ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System

Abstract

Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1β- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.

ADP heptose, a novel pathogen‐associated molecular pattern identified in Helicobacter pylori

“…(9) from the AGS parent cell line [1739; American Type Culture Collection (ATCC), Manassas, VA, USA]. All cells were cultivated at 37°C and 5% CO 2 in Roswell Park Memorial Institute medium (Thermo Fisher Scientific, Waltham, MA) with 10% heat-inactivated fetal calf serum (9). …”

“…Cells were either transfected with β- d -ADP-heptose or β-HBP using lipofectamine 2000 (Thermo Fisher Scientific) under serum-free conditions, treated with sugars without transfection, or left untreated for 3 h at 37°C and 5% CO 2 . To visualize TIFAsome formation, cells were fixed with 4% (w/v) paraformaldehyde and counterstained with DAPI (1:300, H1840-10; Roche) for 2 h. Cover slips were mounted with Mowiol 40–88 (324590; MilliporeSigma, Burlington, MA) and analyzed by laser scanning microscopy using a Leica SP8 confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA) (9). For quantification of TIFAsome-positive cells, 2 images with a ×40 magnification were obtained for every condition.…”

“…As previously described by Zimmermann et al . (9), 1 ng/µl of the isolated RNA was added per reaction, and the final concentration of the primers was 166 nM. qRT-PCR was performed using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) with a 1-step protocol for both RT reaction and PCR reaction.…”

“…More recently, we showed in gastric epithelial cells infected with Helicobacter pylori that the proteins α-kinase 1 (ALPK1) and TRAF-interacting protein with forkhead-associated domain (TIFA) are key components in the response to HBP, leading to activation of canonical NF-κB signaling (9). In this system, too, NF-κB activation was stimulated by HBP upon transfection as well as, presumably, upon translocation to the cytosol via the type-IV secretion system (T4SS) (9). …”

“…The discovery of ALPK1 as an NF-κB pathway–associated signaling molecule (9, 10) and a novel pattern-recognition receptor acting via the rapid formation of TIFAsomes, a previously unknown signaling platform, represent major breakthroughs in the field. However, even in our original study, we were unable to isolate HBP from H. pylori for use in experiments and instead had to rely on chemically synthesized HBP.…”