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.

The actions of IGFs are regulated at various levels. One mechanism involves binding to IGF-binding protein-3 (IGFBP-3) for transport, thus governing bioavailability. IGFBP3 transcription is modulated by many hormones and agents that stimulate or inhibit growth. We have previously shown in pediatric and adult cohorts a correlation between IGFBP-3 serum levels and two single-nucleotide polymorphisms (SNPs) located within the minimal promoter (-202 A/C and -185 C/T). Functionality of these SNPs was further explored in hepatic adenocarcinoma-derived SK-HEP-1 cells using transient transfections of luciferase constructs driven by different haplotypes of the IGFBP3 promoter. Basal luciferase activity revealed a significant haplotype-dependent transcriptional activity (at nucleotides -202 and -185, AC > CC, P < 0.001; AC > CT, P < 0.001; AC > AT, P < 0.001). Insulin treatment produced a similar haplotype dependence of luciferase activity (AC > CC, P = 0.002; AC > CT, P < 0.001; AC > AT, P = 0.011). However, induction ratios (insulin/control) for CC and AT were significantly higher compared with AC and CT (CC > AC, P = 0.03; CC > CT, P = 0.03; AT > AC, P = 0.03; AT > CT, P = 0.04). Gel retardation assays were used to identify upstream stimulatory factor (USF-1 and USF-2) methylation-dependent binding to E-box motifs located between the SNPs. Mutation of the USF binding site resulted in a significant loss of insulin stimulation of luciferase activity in the transfection assay. Chromatin immunoprecipitation with anti-USF-1/-2 showed an enrichment of IGFBP3 promoter in insulin-treated cells compared with unstimulated cells. Bisulfite sequencing of genomic DNA revealed that CpG methylation in the region of USF binding was haplotype dependent. In summary, we report a methylation-dependent USF binding site influencing the basal and insulin-stimulated transcriptional activity of the IGFBP3 promoter.

BMRB accession no. 26852 and PDB 5JBS.

The increasing frequency of antimicrobial resistance is a problem of global importance. Novel strategies are urgently needed to understand and inhibit antimicrobial resistance gene transmission that is mechanistically related to bacterial virulence functions. The conjugative transfer of plasmids by type IV secretion systems is a major contributor to antimicrobial resistance gene transfer. Here, we present a structure-based strategy to identify inhibitors of type IV secretion system-mediated bacterial conjugation. Using differential scanning fluorimetry we screened a fragment library and identified molecules that bind the essential TraE protein of the plasmid pKM101 conjugation machinery. Co-crystallization revealed that fragments bind two alternative sites of the protein and one of them is a novel inhibitor binding site. Based on the structural information on fragment binding we designed novel small molecules that have improved binding affinity. These molecules inhibit the dimerization of TraE, bind to both inhibitor binding sites on TraE and inhibit the conjugative transfer of plasmid pKM101. The strategy presented here is generally applicable for the structure-based design of inhibitors of antimicrobial resistance gene transfer and of bacterial virulence.

Many bacterial pathogens employ multicomponent protein complexes such as type IV secretion systems (T4SSs) to transfer virulence factors into host cells. Here we studied the interaction between two essential T4SS components: the very hydrophobic inner membrane protein VirB6, which may be a component of the translocation channel, and VirB10, which links the inner and outer bacterial membranes. To map the interaction site between these two T4SS components, we conducted alanine scanning and deleted six-amino acid stretches from the N-terminal periplasmic domain of VirB6 from Brucella suis. Using the bacterial two-hybrid system to analyze the effects of these alterations on the VirB6–VirB10 interaction, we identified the amino acid regions 16–21 and 28–33 and Leu-18 in VirB6 as being required for this interaction. SDS-PAGE coupled with Western blotting of cell lysates and native PAGE of detergent-extracted membrane proteins revealed that the corresponding VirB6 residues in Agrobacterium tumefaciens (Phe-20 and amino acids 18–23 and 30–35) modulate the stability of both VirB6 and VirB5. However, the results from immuno-EM and super-resolution microscopy suggested that these regions and residues are not required for membrane association or for polar localization of VirB6. The six-amino acid deletions in the N terminus of VirB6 abolished pilus formation and virulence of A. tumefaciens, and the corresponding deletions in the VirB6 homolog TraD from the plasmid pKM101-T4SS abrogated plasmid transfer. Our results indicate that specific residues of the VirB6 N-terminal domain are required for VirB6 stabilization, its interaction with VirB10, and the incorporation of VirB2 and VirB5 into T-pili.