Oudouhou, F. | bioRxiv Science

bioRxiv2023

Inhibition of the type IV secretion system from antibiotic-resistant Helicobacter pylori clinical isolates supports the potential of Cagα as an anti-virulence target

Helicobacter pylori resistance to antibiotics is a growing problem and it increasingly leads to treatment failure. While the bacterium is present worldwide, the severity of the clinical outcomes is highly dependent on the geographical origin and genetic characteristics of the strains. One of the major virulence factors identified in H. pylori is the cag pathogenicity island (cagPAI), which encodes a type IV secretion used to translocate effectors into human cells. Here, we investigated the genetic variability of the cagPAI among 13 antibiotic-resistant H. pylori strains that were isolated from patient biopsies in Quebec. Seven of the clinical strains carried the cagPAI, but only four could be readily cultivated under laboratory conditions. We observed variability of the sequences of CagA and CagL proteins that are encoded by the cagPAI. All clinical isolates induce interleukin-8 secretion and morphological changes upon co-incubation with epithelial gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cag protein from the model strain H. pylori 26695, reduces interleukin-8 secretion in one of the clinical isolates. Co-incubation with 1G2 also inhibits the assembly of T4SS pili, suggesting a mechanism for its action on T4SS function.

microbiology↗

bioRxiv2018

Fragment-based screening identifies inhibitors of the ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system

Type IV secretion systems are membrane-bound multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system is encoded by the cag pathogenicity island that encodes 27 Cag proteins and most of these are essential for bacterial virulence. We here present our work on the identification and characterization of inhibitors of Cag, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein during thermal denaturation suggesting that they bind Cag. Several of these molecules affect binding of ADP and four of them inhibit the ATPase enzyme activity of Cag. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the ATPase active site. Cross-linking analysis suggests that the active molecules change the conformation of the protein and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cag hexamer. Analysis by X-ray crystallography reveals that molecule 1G2 binds at the interface between Cag subunits. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cag in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins and the identified molecules have potential for the development into antivirulence drugs.

microbiology↗