Christine Josenhans

The Helicobacter pylori cag pathogenicity island ( cag PAI) encodes a complex type IV secretion system (CagT4SS) which is an important virulence factor of H. pylori . Recently, structural detail on the CagT4SS has been substantially improved by cryo-EM. However, important structural and functional information, in particular on protein interactions between T4SS surface proteins, and of T4SS surface proteins with other proteins, is missing. In the present study, we followed the hypothesis that H. pylori T4SS external proteins may form a surface protein assembly, together with other, non-CagT4SS proteins, which may also be essential for T4SS function. Using interaction screens of H. pylori CagT4SS surface proteins, followed by biochemical and functional characterization, we have enhanced the knowledge on protein-protein interactions of CagT4SS extracellular proteins. This also includes newly identified interactions of CagT4SS surface proteins, for instance the VirB2 homolog CagC, the VirB5 homolog CagL and the surface protein CagN of unknown function, with outer membrane proteins HopQ and HopZ. We have further identified and quantitated direct interactions of T4SS surface proteins with outer membrane proteins HopZ and HopQ, which play a role in T4SS functions, and of both HopZ and HopQ with themselves and with host cell factors CEACAM and integrin. Furthermore, we determined an influence of pH on interactions between HopQ/HopZ and CagT4SS components. Utilizing protein tag insertions in H. pylori , we detected surface-exposed association of HopQ and HopZ with T4SS components on bacteria without or with (for HopQ) human gastric epithelial cells. Functionally antagonistic roles of HopQ and HopZ were uncovered in T4SS-dependent early pro- inflammatory human epithelial cell activation. In summary, we identified a network of interactions between H. pylori outer membrane proteins and CagT4SS surface proteins and characterized them as functionally important for transport processes. This will help to refine structural and functional details regarding surface-exposed proteins of the CagT4SS.

Helicobacter pylori genomes contain a large and variable portfolio of methyltransferases (MTases), creating a highly diverse methylome. Here, we characterize a highly conserved ATTAAT-specific MTase, M.Hpy99XIX ( H. pylori strain J99, alternative designations in other strains, M.HpyAVII and M.HpyPVII), the only H. pylori MTase never associated with an endonuclease (“orphan” MTase). Inactivation of M.Hpy99XIX resulted in a significant change in the transcription of >100 genes, despite the fact that only a small subset of their promoter regions contained an ATTAAT target motif. Patterns of transcriptional change showed significant correlations with changes reported for H. pylori mutants in the regulators involved in iron regulation. MTase inactivation also caused a higher susceptibility to diverse metal ions as well as iron chelation and oxidative stress. These phenotypes could be traced back to the methylation of single motifs in the promoter regions of iron transporters frpB1 and fecA1 . Altogether, methylation of individual motifs in promoters can have a large downstream effect, causing major changes to metabolic pathways. These findings suggest that the methylome represents a universal and dynamic interface connecting genome diversity and transcriptional regulation. Very recently, a new ecospecies of H. pylori , Hardy, has been reported. M.Hpy99XIX is present in the majority of “normal” (Ubiquitous) H. pylori strains, whereas no single Hardy strain contained this gene, consistent with other reported differences between Hardy and Ubiquitous strains related to iron/metal homeostasis. ATTAAT methylation is intricately connected with the bacterial transcriptional network, highlighting the important role of bacterial epigenetic modifications in bacterial physiology and pathogenesis. IMPORTANCE: Helicobacter pylori has one of the largest repertoires of methyltransferases. Methylation has been associated with multiple functions in H. pylori , including the defense against foreign DNA and transcriptional regulation. Regulation of gene expression by methylation has the potential to influence many distant genes across the genome via target motifs in proximity to transcription start sites. Here, we sought to understand the role of M.Hpy99XIX, an orphan methyltransferase targeting the ATTAAT motif that is highly conserved in H. pylori . We show that by directly regulating specific genes involved in iron uptake via methylated ATTAAT motifs, M.Hpy99XIX has a significant effect on iron homeostasis by triggering the canonical iron regulatory pathway. Furthermore, we show that M.Hpy99XIX appears to have been acquired after the split between the two ecospecies of H. pylori , suggesting that its role in the tuning of iron homeostasis might have contributed to this divergence.

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a complex virulence-associated type IV secretion system (CagT4SS). Recently, structural detail on the CagT4SS has been substantially improved by Cryo-EM. However, important structural and functional information is still missing. In the present study, we followed the hypothesis that H. pylori T4SS external proteins may form a surface-exposed assembly, together with non-CagT4SS proteins, which may be essential for T4SS function. Using interaction screens followed by biochemical and functional characterization, we have enhanced the knowledge about functional protein–protein interactions of the CagT4SS extracellular proteins. This comprises newly identified interactions of CagT4SS surface proteins, including the VirB2 homolog CagC, the VirB5 homolog CagL and CagN, with outer membrane proteins HopQ and HopZ. We have further quantitated direct, pH dependent, interactions of T4SS surface proteins with HopZ and HopQ, with host cell factors CEACAM and integrin, and self-interactions of both HopZ and HopQ. Utilizing chromosomal tag insertions in H. pylori, we detected surface-exposed colocalization of HopQ with T4SS components in the absence or, for HopQ, also in the presence of human gastric epithelial cells. Functionally antagonistic roles of HopQ and HopZ were uncovered in early proinflammatory human epithelial cell activation by the T4SS. In summary, we identified a network of interactions between H. pylori outer membrane proteins and CagT4SS surface proteins that are functionally relevant for T4SS-dependent transport processes. This study provides a valuable resource guiding future studies to refine structure and mechanistic roles of the surface-exposed portions of the CagT4SS.

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.

Bacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogen Helicobacter pylori , heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis.

Heptose metabolites including ADP-heptose are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent pro-inflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question, how pro-inflammatory ADP-heptose biosynthesis can be regulated by the bacteria. We have characterized the inter-strain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori , which impact cell-autonomous pro-inflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains, and was also strain-variably expressed. Transcript amounts of genes in the hldE gene cluster displayed intra-strain and inter-strain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori LPS heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE and GmhB proteins. On the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry, the structures of major reaction products were identified as β-D-ADP-heptose and β-heptose-1-monophosphate. A pro-inflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed to uncover additional strain variation in generating heptose metabolites.

Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and triggers TIFA phosphorylation on threonine 9. This leads to the assembly of large TIFA oligomers called TIFAsomes, activation of NF-κB and pro-inflammatory gene expression. Furthermore, mutations in ALPK1 are associated with inflammatory syndromes and cancers. While this kinase is of growing medical interest, only few tools are available to characterize its activity. Here, we describe a versatile non-radioactive ALPK1 in vitro kinase assay based on protein thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are also weakly phosphorylated by ALPK1. Interestingly, we find that ALPK1 itself is phosphorylated in response to ADPH recognition during Shigella flexneri and Helicobacter pylori infection and that disease-associated ALPK1 mutants exhibit altered kinase activity. In particular, T237M and V1092A mutations associated with ROSAH syndrome and spiradenoma/spiradenocarcinoma respectively, exhibit enhanced ADPH-induced kinase activity and constitutive assembly of TIFAsomes. Altogether, this study provides new insights into the ADPH sensing pathway and a new tool for measuring the activity of ALPK1.