Helicobacter pylori CagA protein is a potent and broad-spectrum amyloid inhibitor

Abstract

Bacteria, the smallest and most abundant life forms on Earth, have been a source of insights that have had a considerable impact on human health. Helicobacter pylori has captured substantial attention due to its role in provoking an array of gastrointestinal ailments and other human diseases. Here, we report that H. pylori releases the protein CagA (cytotoxin-associated gene A) that strongly inhibits formation of both functional (bacterial biofilm) and pathogenic amyloid assemblies by targeting various stages during fibril formation. CagA's broad substrate specificity reveals a mechanism whereby H. pylori interferes with other bacteria and humans, offering approaches to combat bacterial infections and human protein misfolding diseases.

Fig. 1.. Physiology, structural organization, and recombinant preparation of H. pylori CagA.

(A) H. pylori colonizes the human stomach lining and has been associated with various human diseases, including gastrointestinal disorders, cardiovascular diseases, as well as neurodegenerative disorders like AD and PD. Created in BioRender. Chen, G. (2025) https://BioRender.com/v27f352. (B) CagA consists of a N-terminal (1 to 884) and a C-terminal region (885 to 1186). The C-terminal region is disordered, and the N-terminal region can be further subdivided into three distinct sections: domain I (D1), domain II (D2), and domain III (D3) (Protein Data Bank ID 4DVY). Domain I comprises 10 α helices, domain II features a substantial antiparallel β sheet, including a subdomain with five α helices and two short β sheets, while domain III adopts a four-helix bundle structure. (C) The N-terminal region, CagA^N, was expressed in E. coli and purified using immobilized metal affinity chromatography (IMAC), ion exchange chromatography (IEC), and size exclusion chromatography (SEC). SDS–polyacrylamide gel electrophoresis (PAGE) analysis indicated high purity. (D) Circular dichroism (CD) was used to measure the secondary structure, revealing an α helix dominant conformation. MRE, mean residue ellipticity.

Fig. 2.. CagA^N prevents bacterial biofilm formation and functional amyloid assembly.

(A) Schematic presentation of bacterial functional amyloid and biofilm formation, where functional amyloid is the structure scaffold of biofilm. (B) CagA^N inhibits Pseudomonas biofilm formation. Pseudomonas sp. UK4 (WT and pFap species) were incubated with and without recombinant CagA^N proteins, and the biofilm formation was evaluated by Gram’s crystal violet staining. For both WT and pFap species, CagA^N showed dose-dependent effects on the reducing biofilm formation. The data point for the effect of 0.3 μM CsgA on UK4WT was excluded as it was identified as an outlier. (C and D) Visualization of the biofilm in the presence or absence of different concentrations of CagA^N with confocal scanning laser microscopy. Red color is for dead bacteria (TOTO-3), blue is for live bacteria (SYTO-41), and green is for amyloid (AmyGreen). (E and F) Pseudomonas secretes FapC protein that assembles into amyloid fibrils. The inhibition effects of CagA^N on FapC fibril formation were studied by ThT assay with 15 μM FapC and different concentrations of CagA^N from 0 to 1280 nM. The term “mass” here refers to the relative amount of amyloid fibrils formed, which correlates with the ThT fluorescence signal. To transform the ThT fluorescence data into a relative “fibril mass fraction,” we normalized the fluorescence intensity to the maximum value observed in each experiment, thereby representing the progression of fibril formation as a fraction of the total fibril mass formed under the experimental conditions. (G) The half time (𝜏_1/2) was extracted by sigmoidal fitting of the dataset in (F). The error bar stands for the SD. Created in BioRender. Chen, G. (2025) https://BioRender.com/j64n375.

Fig. 3.. CagA prevents pathogenic amyloid formation, associated with AD, PD, and T2D.

(A) Schematic presentation of pathogenic amyloid formation and some of the relevant human diseases. Created in BioRender. Chen, G. (2025) https://BioRender.com/c15w610. (B) CagA’s inhibition of Aβ42 fibril formation was tested by ThT assay with 3 μM Aβ42 and 0 to 20 nM CagA^N. α-syn, α-synuclein. (C) The 𝜏_1/2 was extracted by sigmoidal fitting of the dataset in (B). (D) CagA’s inhibition of human disease associated amyloidogenic proteins and peptides with different size and charges, including 5 μM Aβ40 (AD relevant, negatively charged, 4 kDa) versus 0 to 1 nM CagA^N, 20 μM tau (AD relevant, positively charged, 46 kDa) versus 0 to 1000 nM CagA^N, 30 μM α-synuclein (PD relevant, negatively charged, 14.4 kDa) versus 0 to 500 nM CagA^N, and 10 μM islet amyloid polypeptide (IAPP) (T2D relevant, positively charged, 4 kDa) versus 0 to 100 nM CagA^N. The τ_1/2 values were extracted by sigmoidal fitting of the dataset in fig. S1. (E) The inhibition effects of CagA^N on 30 μM α-synuclein fibril formation at pH 5.5 were tested by ThT assay with CagA^N concentration from 0 to 1 μM. The 𝜏_1/2 was extracted by sigmoidal fitting of the dataset in fig. S2. (F) Canonical chaperone activity evaluation of CagA. Kinetics of aggregation of 600 nM citrate synthase (CS) at 45°C alone (purple), in the presence of 0.6 μM CagA^N (pink), 1.2 μM CagA^N (red), or 2.4 μM CagA^N (dark red). The error bar stands for the SD. a.u., arbitrary unit.

Fig. 4.. CagA^N interferes with different microscopic events of the fibril formation process of various amyloid peptides or proteins.

The kinetics of amyloid aggregation involves distinct stages (43): In the primary nucleation phase, monomers come together to create a nucleus (k_n), from which a fibril can start to elongate (k₊); simultaneously, during secondary nucleation (k₂), monomers adhere to the fibril’s surface, catalyzing the development of a new nucleus and facilitating exponential fibril growth. Refer to the schematic representation in (A) and (B) for a visual depiction of this process. (A) CagA^N suppresses amyloid formation of different bacterial functional amyloid proteins, i.e., CsgA from E. coli and FapC from Pseudomonas. For these amyloidogenic proteins, CagA^N predominantly obstructs the elongation process, as indicated by a red cross. (B) CagA^N impedes the formation of amyloid fibrils by various human pathogenic amyloid peptides or proteins through diverse mechanisms. In the case of the AD-associated Aβ42 and Aβ40 peptides, mainly primary nucleation is blocked as the oligomers are likely stabilized by the CagA proteins. Conversely, for the AD-relevant protein tau, both secondary nucleation and elongation are affected by CagA^N. Regarding the PD-related protein α-synuclein, CagA^N inhibits the elongation process. The IAPP peptide, associated with T2D, has its fibrillization inhibited by CagA^N, affecting both secondary nucleation and elongation processes. The details of global fitting for each dataset are included in figs. S2 to S4. α-syn, α-synuclein. Created in BioRender. Chen, G. (2025) https://BioRender.com/m37r047.

Fig. 5.. The interaction of CagA^N with amyloidogenic substrates.

(A) NMR spectrum of ¹⁵N-labeled α-synuclein monomer with (red) and without CagA^N (blue). (B) The relative intensity of each amino acid of α-synuclein with and without CagA^N. Residues marked with an asterisk (*) were excluded from the analysis due to too low signal-to-noise ratio. I/I₀ is the relative NMR signal intensity with and without CagA^N. (C and D) Surface plasmon resonance (SPR) analysis of CagA^N and α-synuclein oligomers or fibrils. (E) NMR spectrum of ¹⁵N-labeled Aβ42 monomer with (red) and without CagA^N (blue). (F) The relative intensity of each amino acid of Aβ42 with and without CagA^N. Residues marked with an asterisk (*) were excluded from the analysis due to too low signal-to-noise ratio. (G) AlphaFold 3 prediction of the interactions of CagA^N and different amyloid peptides or proteins, i.e., Aβ42 (four subunits, mimicking the aggregates) and α-synuclein (six subunits, mimicking the fibrils). (H) The inhibition effects of CagA^N domain II on 3 μM Aβ42 fibril formation was tested by ThT assay with CagA^N concentration from 0 to 100 nM. (I) The τ_1/2 and r_max were extracted by sigmoidal fitting of the dataset in (H). The error bar stands for the SD. (J) Electrospray ionization mass spectrometry (ESI-MS) spectra of the CagA^N domain II at pH 8.0. The charge states and the molecular weight are indicated. D1, domain I; D2, domain II; D3, domain III; α-syn, α-synuclein; ppm, parts per million; m/z, mass/charge ratio.