Bianca Bauer

The Human Gastric Pathogen Helicobacter pylori and Its Association with Gastric Cancer and Ulcer Disease

With the momentous discovery in the 1980s that a bacterium, Helicobacter pylori, can cause peptic ulcer disease and gastric cancer, antibiotic therapies and prophylactic measures have been successful, only in part, in reducing the global burden of these diseases. To date, ~700,000 deaths worldwide are still attributable annually to gastric cancer alone. Here, we review H. pyloris contribution to the epidemiology and histopathology of both gastric cancer and peptic ulcer disease. Furthermore, we examine the host-pathogen relationship and H. pylori biology in context of these diseases, focusing on strain differences, virulence factors (CagA and VacA), immune activation and the challenges posed by resistance to existing therapies. We consider also the important role of host-genetic variants, for example, in inflammatory response genes, in determining infection outcome and the role of H. pylori in other pathologies—some accepted, for example, MALT lymphoma, and others more controversial, for example, idiopathic thrombocytic purpura. More recently, intriguing suggestions that H. pylori has protective effects in GERD and autoimmune diseases, such as asthma, have gained momentum. Therefore, we consider the basis for these suggestions and discuss the potential impact for future therapeutic rationales.

Temporal resolution of two-tracked NF-kappa B activation by Legionella pneumophila

The intracellular pathogen Legionella pneumophila activates the transcription factor NF‐κB in macrophages and human epithelial cells, contributing to cytokine production and anti‐apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF‐κB by L. pneumophila in human epithelial cells, using a p65‐GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin‐deficient (ΔfliA and ΔflaA) mutants could not induce this first, TLR5 and MyD88‐dependent activation. The second p65 translocation event, however, is a long‐term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65‐GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IκBα and upregulation of anti‐apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (ΔdotA; ΔicmB/dotO), Dot/Icm effectors (ΔsdbA; ΔlubX) and two bacterial effector mutants (ΔenhC; ΔptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF‐κB activation and intracellular replication and hints at an active interference of NF‐κB signalling by L. pneumophila.

Helicobacter pylori outer membrane protein HopQ identified as a novel T4SS‐associated virulence factor

Helicobacter pylori is a bacterial pathogen that colonizes the gastric niche of ∼ 50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)‐encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large‐scale screen of an H. pylori transposon mutant library using activation of the pro‐inflammatory transcription factor NF‐κB in human gastric epithelial cells as a measure of T4SS function. Analysis of ∼ 3000 H. pylori mutants revealed three non‐cagPAI genes that affected NF‐κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA‐mediated host cellresponses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS‐dependent activation of NF‐κB, induction of MAPK signalling and secretion of interleukin 8 (IL‐8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non‐cagPAI‐encoded cofactor of T4SS function.

Helicobacter pylori HP0518 affects flagellin glycosylation to alter bacterial motility

Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as gastric cancer. Mounting evidence suggests this pathogens motility is prerequisite for successful colonization of human gastric tissues. Here, we isolated an H. pylori G27 HP0518 mutant exhibiting altered motility in comparison to its parental strain. We show that the mutants modulated motility is linked to increased levels of O‐linked glycosylation on flagellin A (FlaA) protein. Recombinant HP0518 protein decreased glycosylation levels of H. pylori flagellin in vitro, indicating that HP0518 functions in deglycosylation of FlaA protein. Furthermore, mass spectrometric analysis revealed increased glycosylation of HP0518 FlaA was due to a change in pseudaminic acid (Pse) levels on FlaA; HP0518 mutant‐derived flagellin contained approximately threefold more Pse than the parental strain. Further phenotypic and molecular characterization demonstrated that the hyper‐motile HP0518 mutant exhibits superior colonization capabilities and subsequently triggers enhanced CagA phosphorylation and NF‐κB activation in AGS cells. Our study shows that HP0518 is involved in the deglycosylation of flagellin, thereby regulating pathogen motility. These findings corroborate the prominent function of H. pylori flagella in pathogen–host cell interactions and modulation of host cell responses, likely influencing the pathogenesis process.

High-throughput and single-cell imaging of NF-κB oscillations using monoclonal cell lines

BackgroundThe nuclear factor-κB (NF-κB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-κB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-κB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-κB-induced genes is regulated by differential dynamics of single NF-κB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-κB activation have been observed in response to the cytokine tumor necrosis factor α (TNFα), little is known about the occurrence of oscillations in response to bacterial infections.ResultsTo quantitatively assess NF-κB dynamics we generated human and murine monoclonal cell lines that stably express the NF-κB subunit p65 fused to GFP. Furthermore, a high-throughput assay based on automated microscopy coupled to image analysis to quantify p65-nuclear translocation was established. Using this assay, we demonstrate a stimulus- and cell line-specific temporal control of p65 translocation, revealing, for the first time, oscillations of p65 translocation in response to bacterial infection. Oscillations were detected at the single-cell level using real-time microscopy as well as at the population level using high-throughput image analysis. In addition, mathematical modeling of NF-κB dynamics during bacterial infections predicted masking of oscillations on the population level in asynchronous activations, which was experimentally confirmed.ConclusionsTaken together, this simple and cost effective assay constitutes an integrated approach to infer the dynamics of NF-κB kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-κB can be identified and analyzed, providing new possibilities for a wide range of applications from therapeutic discovery and understanding of disease to host-pathogen interactions.

Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

ABSTRACT Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.

Differential Expression of Human Beta Defensin 2 and 3 in Gastric Mucosa of Helicobacter pylori-Infected Individuals

Antimicrobial peptides are key players of initial innate immune responses to human pathogens. Two major representatives, the human beta defensin 2 and 3 (hBD2 and hBD3), are both known to be regulated by, and to affect viability of, Helicobacter pylori. Previously, it was demonstrated in vitro that H. pylori actively abrogates hBD3 expression during prolonged infections. Here, we comprehensively assessed hBD2 and hBD3 expression ex vivo in the gastric mucosa of healthy individuals.

H. pylori selectively blocks EGFR endocytosis via the non‐receptor kinase c‐Abl and CagA

Helicobacter pylori infection is a primary cause of peptic ulcers and is associated with gastric carcinogenesis. The H. pylori‐induced pathophysiology may be linked to the deregulation of EGFR signalling. Elevated mucosal levels of EGF and the EGFR have been found in antral gastric biopsies of H. pylori‐infected patients. A critical mechanism for regulating EGFR signalling is ligand‐induced endocytosis. The internalized receptor recycles back to the plasma membrane for continued signalling or is targeted for degradation terminating receptor signalling. Here, we show that H. pylori blocks EGFR endocytosis and receptor degradation upon prolonged infection of gastric epithelial cells. Moreover, this inhibition occurs via a CagA‐dependent, but CagA phosphorylation‐independent activation of the non‐receptor kinase c‐Abl, which in turn phosphorylates the EGFR target site pY1173. This suggests a novel CagA‐induced host cell response that is independent of CagA tyrosine phosphorylation. Our data indicate an intriguing strategy of H. pylori in host cell manipulations by altering selective receptor populations via a CagA‐dependent endocytic mechanism. Furthermore, we identified a new role for c‐Abl in phosphorylation of the EGFR target site pY1173 during H. pylori infection.

Chlamydia trachomatis infection prevents front–rear polarity of migrating HeLa cells

Chlamydiae are obligate intracellular bacterial pathogens that cause trachoma, sexually transmitted diseases and respiratory infections in humans. Fragmentation of the host cell Golgi apparatus (GA) is essential for chlamydial development, whereas the consequences for host cell functions, including cell migration are not well understood. We could show that Chlamydia trachomatis‐infected cells display decelerated migration and fail to repopulate monolayer scratch wounds. Furthermore, infected cells lost the ability to reorient the fragmented GA or the microtubule organization centre (MTOC) after a migratory stimulus. Silencing of golgin‐84 phenocopied this defect in the absence of the infection. Interestingly, GA stabilization via knockdown of Rab6A and Rab11A improved its reorientation in infected cells and it was fully rescued after inhibition of Golgi fragmentation with WEHD‐fmk. These results show that C. trachomatis infection perturbs host cell migration on multiple levels, including the alignment of GA and MTOC.

RNA-interference based screen identifies new factors important for NF-kappaB activation and termination

The transcription factor NF-kappaB is a key mediator of the innate immune system. Although tremendous research efforts over the past decades have led to a more and more detailed understanding of NF-kappaB signaling, there are still missing pieces in the puzzle, especially upstream of the IKK complex and in the termination of the signaling. To identify more of the factors important for this signaling pathway we have conducted an RNA-interference based screen. For this purpose, we have developed an assay for high throughput analysis using a human epithelial cell line stably expressing a p65-GFP-fusion construct. The nuclear translocation of p65-GFP can be quantified by automated microscopic analysis. Three different stimuli were compared: the cytokines TNF-alpha and IL1-beta and the gastric pathogen Helicobacter pylori. We chose H. pylori as inducer because permanent infection with this bacterium can lead to chronic inflammation, ulceration and cancerogenesis and NF-kappaB is thought to be crucial in the promotion of this pathology. Furthermore, using different time points of the activation, we screened not only for factors important for activation, but also for termination of the signal. In terms of activation, the screen identified known factors like IKKalpha and IKKbeta as well as factors so far not linked to the NF-kappaB pathway. Interestingly, two factors were identified that are specific for NF-kappaB activation after H. pylori infection and not necessary for NF-kappaB activation by the cytokines TNFalpha or IL-1beta. Regarding termination, the screen identified among other factors an ubiquitin E3-Ligase so far not linked to the pathway. Upon down-regulation of this E3-Ligase, p65-GFP resides longer in the nucleus. This correlates with a strong degradation of IkappaBalpha. The screen was conducted with a library of siRNAs against 646 kinases and associated proteins, and is currently expanded to a genome wide scale.