Metz, F. | bioRxiv Science

bioRxiv2025

Functional and intricate interaction network connecting Helicobacter pylori Cag Type 4 Secretion System surface proteins with outer membrane proteins HopQ and HopZ

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a complex type IV secretion system (CagT4SS) which is an important virulence factor of H. pylori. Recently, structural detail on the CagT4SS has been substantially improved by cryo-EM. However, important structural and functional information, in particular on protein interactions between T4SS surface proteins, and of T4SS surface proteins with other proteins, is missing. In the present study, we followed the hypothesis that H. pylori T4SS external proteins may form a surface protein assembly, together with other, non-CagT4SS proteins, which may also be essential for T4SS function. Using interaction screens of H. pylori CagT4SS surface proteins, followed by biochemical and functional characterization, we have enhanced the knowledge on protein-protein interactions of CagT4SS extracellular proteins. This also includes newly identified interactions of CagT4SS surface proteins, for instance the VirB2 homolog CagC, the VirB5 homolog CagL and the surface protein CagN of unknown function, with outer membrane proteins HopQ and HopZ. We have further identified and quantitated direct interactions of T4SS surface proteins with outer membrane proteins HopZ and HopQ, which play a role in T4SS functions, and of both HopZ and HopQ with themselves and with host cell factors CEACAM and integrin. Furthermore, we determined an influence of pH on interactions between HopQ/HopZ and CagT4SS components. Utilizing protein tag insertions in H. pylori, we detected surface-exposed association of HopQ and HopZ with T4SS components on bacteria without or with (for HopQ) human gastric epithelial cells. Functionally antagonistic roles of HopQ and HopZ were uncovered in T4SS-dependent early pro- inflammatory human epithelial cell activation. In summary, we identified a network of interactions between H. pylori outer membrane proteins and CagT4SS surface proteins and characterized them as functionally important for transport processes. This will help to refine structural and functional details regarding surface-exposed proteins of the CagT4SS.

microbiology↗

bioRxiv2023

Helicobacter pylori modulates heptose metabolite biosynthesis and heptose-dependent innate immune host cell activation by multiple mechanisms

Heptose metabolites including ADP-heptose are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent pro-inflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question, how pro-inflammatory ADP-heptose biosynthesis can be regulated by the bacteria. We have characterized the inter-strain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous pro-inflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains, and was also strain-variably expressed. Transcript amounts of genes in the hldE gene cluster displayed intra-strain and inter-strain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori LPS heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE and GmhB proteins. On the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry, the structures of major reaction products were identified as {beta}-D-ADP-heptose and {beta}-heptose-1-monophosphate. A pro-inflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed to uncover additional strain variation in generating heptose metabolites.

microbiology↗