Biology subjects

Josenhans, C.

Publications and source records attributed to Josenhans, C..

5 recordsLinked to original sources

bioRxiv2025

Functional and intricate interaction network connecting Helicobacter pylori Cag Type 4 Secretion System surface proteins with outer membrane proteins HopQ and HopZ

The Helicobacter pylori cag pathogenicity island (cagPAI) 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.

microbiology↗

bioRxiv2024

The Helicobacter pylori orphan ATTAAT-specific methyltransferase M.Hpy99XIX plays a central role in the coordinated regulation of genes involved in iron metabolism

H. 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, 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 ArsRS regulators involved in iron regulation. The 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 Hardy of H. pylori 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.

microbiology↗

bioRxiv2023

Helicobacter pylori modulates heptose metabolite biosynthesis and heptose-dependent innate immune host cell activation by multiple mechanisms

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 {beta}-D-ADP-heptose and {beta}-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.

microbiology↗

bioRxiv2023

In vitro ALPK1 kinase assay reveals new insights into ADP-heptose sensing pathway and kinase activity of disease-associated ALPK1 mutants

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-{kappa}B 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.

immunology↗

bioRxiv2018

The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori

Helicobacter pylori encodes a large number of Restriction-Modification (R-M) systems despite its small genome.R-M systems have been described as \"primitive immune systems\" in bacteria, but the role of methylation in bacterial gene regulation and other processes is increasingly accepted. Every H.pylori strain harbours a unique set of R-M systems resulting in a highly diverse methylome. We identified a highly conserved GCGC-specific m5C MTase (JHP1050) that was predicted to be active in all of 459 H.pylori genome sequences analyzed. Transcriptome analysis of two H.pylori strains and their respective MTase mutants showed that inactivation of the MTase led to changes in the expression of 225 genes in strain J99, and 29 genes in strain BCM-300.10 genes were differentially expressed in both mutated strains. Combining bioinformatic analysis and site-directed mutagenesis, we demonstrated that motifs overlapping the promoter influence the expression of genes directly, while methylation of other motifs might cause secondary effects.Thus, m5C methylation modifies the transcription of multiple genes, affecting important phenotypic traits that include adherence to host cells, natural competence for DNA uptake, bacterial cell shape, and susceptibility to copper.

microbiology↗