Single-base resolution quantitative genome methylation analysis in the model bacterium Helicobacter pylori by enzymatic methyl sequencing (EM-Seq) reveals influence of strain, growth phase, and methyl homeostasis.

BACKGROUND: Bacterial epigenetics is a rapidly expanding research field. DNA methylation by diverse bacterial methyltransferases (MTases) contributes to genomic integrity and replication, and many recent studies extended MTase function also to global transcript regulation and phenotypic variation. Helicobacter pylori is currently one of those bacterial species which possess the highest number and the most variably expressed set of DNA MTases. Next-generation sequencing technologies can directly detect DNA base methylation. However, they still have limitations in their quantitative and qualitative performance, in particular for cytosine methylation. RESULTS: As a complementing approach, we used enzymatic methyl sequencing (EM-Seq), a technology recently established that has not yet been fully evaluated for bacteria. Thereby, we assessed quantitatively, at single-base resolution, whole genome cytosine methylation for all methylated cytosine motifs in two different H. pylori strains and isogenic MTase mutants. EM-Seq reliably detected both m5C and m4C methylation. We demonstrated that three different active cytosine MTases in H. pylori provide considerably different levels of average genome-wide single-base methylation, in contrast to isogenic mutants which completely lost specific motif methylation. We found that strain identity and changed environmental conditions, such as growth phase and interference with methyl donor homeostasis, significantly influenced quantitative global and local genome-wide methylation in H. pylori at specific motifs. We also identified significantly hyper- or hypo-methylated cytosines, partially linked to overlapping MTase target motifs. Notably, we revealed differentially methylated cytosines in genome-wide coding regions under conditions of methionine depletion, which can be linked to transcript regulation. CONCLUSIONS: This study offers new knowledge on H. pylori global and local genome-wide methylation and establishes EM-Seq for quantitative single-site resolution analyses of bacterial cytosine methylation.

Identification of Antimotilins, Novel Inhibitors of Helicobacter pylori Flagellar Motility That Inhibit Stomach Colonization in a Mouse Model.

New treatment options against the widespread cancerogenic gastric pathogen Helicobacter pylori are urgently needed. We describe a novel screening procedure for inhibitors of H. pylori flagellar biosynthesis. The assay is based on a flaA flagellin gene-luciferase reporter fusion in H. pylori and was amenable to multi-well screening formats with an excellent Z factor. We screened various compound libraries to identify virulence blockers ("antimotilins") that inhibit H. pylori motility or the flagellar type III secretion apparatus. We identified compounds that either inhibit both motility and the bacterial viability, or the flagellar system only, without negatively affecting bacterial growth. Novel anti-virulence compounds which suppressed flagellar biosynthesis in H. pylori were active on pure H. pylori cultures in vitro and partially suppressed motility directly, reduced flagellin transcript and flagellin protein amounts. We performed a proof-of-principle treatment study in a mouse model of chronic H. pylori infection and demonstrated a significant effect on H. pylori colonization for one antimotilin termed Active2 even as a monotherapy. The diversity of the intestinal microbiota was not significantly affected by Active2. In conclusion, the novel antimotilins active against motility and flagellar assembly bear promise to complement commonly used antibiotic-based combination therapies for treating and eradicating H. pylori infections. IMPORTANCE Helicobacter pylori is one of the most prevalent bacterial pathogens, inflicting hundreds of thousands of peptic ulcers and gastric cancers to patients every year. Antibacterial treatment of H. pylori is complicated due to the need of combining multiple antibiotics, entailing serious side effects and increasing selection for antibiotic resistance. Here, we aimed to explore novel nonantibiotic approaches to H. pylori treatment. We selected an antimotility approach since flagellar motility is essential for H. pylori colonization. We developed a screening system for inhibitors of H. pylori motility and flagellar assembly, and identified numerous novel antibacterial and anti-motility compounds (antimotilins). Selected compounds were further characterized, and one was evaluated in a preclinical therapy study in mice. The antimotilin compound showed a good efficacy to reduce bacterial colonization in the model, such that the antimotilin approach bears promise to be further developed into a therapy against H. pylori infection in humans.

Fusion of Bacterial Flagellin to a Dendritic Cell-Targeting αCD40 Antibody Construct Coupled With Viral or Leukemia-Specific Antigens Enhances Dendritic Cell Maturation and Activates Peptide-Responsive T Cells.

Conventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens ex vivo, suffer biological issues such as impaired DC migration capacity and laborious GMP production procedures. In a promising alternative, antigens are targeted to DC-associated endocytic receptors in vivo with antibody-antigen conjugates co-administered with toll-like receptor (TLR) agonists as adjuvants. To combine the potential advantages of in vivo targeting of DCs with those of conjugated TLR agonists, we generated a multifunctional antibody construct integrating the DC-specific delivery of viral- or tumor-associated antigens and DC activation by TLR ligation in one molecule. We validated its functionality in vitro and determined if TLR ligation might improve the efficacy of such a molecule. In proof-of-principle studies, an αCD40 antibody containing a CMV pp65-derived peptide as an antigen domain (αCD40CMV) was genetically fused to the TLR5-binding D0/D1 domain of bacterial flagellin (αCD40.FlgCMV). The analysis of surface maturation markers on immature DCs revealed that fusion of flagellin to αCD40CMV highly increased DC maturation (3.4-fold elevation of CD80 expression compared to αCD40CMV alone) by specifically interacting with TLR5. Immature DCs loaded with αCD40.FlgCMV induced significantly higher CMVNLV-specific T cell activation and proliferation compared to αCD40CMV in co-culture experiments with allogeneic and autologous T cells (1.8-fold increase in % IFN-γ/TNF-α+ CD8+ T cells and 3.9-fold increase in % CMVNLV-specific dextramer+ CD8+ T cells). More importantly, we confirmed the beneficial effects of flagellin-dependent DC stimulation using a tumor-specific neoantigen as the antigen domain. Specifically, the acute myeloid leukemia (AML)-specific mutated NPM1 (mNPM1)-derived neoantigen CLAVEEVSL was delivered to DCs in the form of αCD40mNPM1 and αCD40.FlgmNPM1 antibody constructs, making this study the first to investigate mNPM1 in a DC vaccination context. Again, αCD40.FlgmNPM1-loaded DCs more potently activated allogeneic mNPM1CLA-specific T cells compared to αCD40mNPM1. These in vitro results confirmed the functionality of our multifunctional antibody construct and demonstrated that TLR5 ligation improved the efficacy of the molecule. Future mouse studies are required to examine the T cell-activating potential of αCD40.FlgmNPM1 after targeting of dendritic cells in vivo using AML xenograft models.