Biology subjects

Sygusch, J.

Publications and source records attributed to Sygusch, J..

3 recordsLinked to original sources

bioRxiv2024

Inner membrane components of the plasmid pKM101 type IV secretion system TraE and TraD are DNA-binding proteins

The increase of antimicrobial resistance constitutes a significant threat to human health. One of the mechanisms responsible for the spread of resistance to antimicrobials is the transfer of plasmids between bacteria by conjugation. This process is mediated by type IV secretion systems (T4SS) and previous studies have provided in vivo evidence for interactions between DNA and components of the T4SS. Here, we purified TraD and TraE, two inner membrane proteins from the Escherichia coli pKM101 T4SS. Using electrophoretic mobility shift assays and fluorescence polarization we showed that the purified proteins both bind single-stranded and double-stranded DNA in the nanomolar affinity range. The previously identified conjugation inhibitor BAR-072 inhibits TraE DNA binding in vitro, providing evidence for its mechanism of action. Site-directed mutagenesis identified conserved amino acids that are required for conjugation that may be targets for the development of more potent conjugation inhibitors.

biochemistry↗

bioRxiv2023

Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the{square}cag{square}pathogenicity island, and we chose Cag, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cag in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cag subunits and mutagenesis of the binding site identified Cag residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889 and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cag, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

biochemistry↗

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↗