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 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 system (T4SS) 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 Québec. 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 gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cagα 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.

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

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, 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 suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. 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 Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.

Analysis of Novel Interactions between Components of the Selenocysteine Biosynthesis Pathway, SEPHS1, SEPHS2, SEPSECS, and SECp43.

In mammalian cells, the incorporation of the 21st amino acid, selenocysteine, into proteins is guided by the Sec machinery. The function of this protein complex requires several protein-protein and protein-RNA interactions, leading to the incorporation of selenocysteine at UGA codons. It is guided by stem-loop structures localized in the 3' untranslated regions of the selenoprotein-encoding genes. Here, we conducted a global analysis of interactions between the Sec biosynthesis and incorporation components using a bioluminescence resonance energy transfer assay in mammalian cells that showed that selenocysteine synthase (SEPSECS), SECp43, and selenophosphate synthetases SEPHS1 and SEPHS2 form oligomers in eukaryotic cells. We also showed that SEPHS2 interacts with SEPSECS and SEPHS1; these interactions were confirmed by co-immunoprecipitation. To further analyze the interactions of SECp43, the protein was expressed in Escherichia coli, and small-angle X-ray scattering analysis revealed that it is a globular protein comprising two RNA-binding domains. Using phage display, we identified potential interaction sites and highlighted two residues (K166 and P167) required for its dimerization. The SECp43 structural model presented here constitutes the basis of future exploration of the protein-protein interactions among early components of the selenocysteine biosynthesis and incorporation pathway.

Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.