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"Gerhard, Markus"
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Identification and characterization of nucleobase-modified aptamers by click-SELEX
Aptamers are single-stranded oligonucleotides that are in vitro-selected to recognize their target molecule with high affinity and specificity. As they consist of the four canonical nucleobases, their chemical diversity is limited, which in turn limits the addressable target spectrum. Introducing chemical modifications into nucleic acid libraries increases the interaction capabilities of the DNA and thereby the target spectrum. Here, we describe a protocol to select nucleobase-modified aptamers by using click chemistry (CuAAC) to introduce the preferred chemical modification. The use of click chemistry to modify the DNA library enables the introduction of a wide range of possible functionalities, which can be customized to the requirements of the target molecule and the desired application. This protocol yields modified DNA aptamers with extended interaction properties that are not accessible with the canonical set of nucleotides. After synthesis of the starting library containing a commercially available, alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU)) instead of thymidine, the library is functionalized with the modification of choice by CuAAC. The thus-modified DNA is incubated with the target molecule and the best binding sequences are recovered. The chemical modification is removed during the amplification process. Therefore, this protocol is compatible with conventional amplification procedures and avoids enzymatic incompatibility problems associated with more extensive nucleobase modifications. After single-strand generation, the modification is reintroduced into the enriched library, which can then be subjected to the subsequent selection cycle. The duration of each selection cycle as outlined in the protocol is â^¼1 d.
Journal Article
Effects of Helicobacter pylori infection on intestinal microbiota, immunity and colorectal cancer risk
Infecting about half of the world´s population, Helicobacter pylori is one of the most prevalent bacterial infections worldwide and the strongest known risk factor for gastric cancer. Although H. pylori colonizes exclusively the gastric epithelium, the infection has also been associated with various extragastric diseases, including colorectal cancer (CRC). Epidemiological studies reported an almost two-fold increased risk for infected individuals to develop CRC, but only recently, direct causal and functional links between the chronic infection and CRC have been revealed. Besides modulating the host intestinal immune response, H. pylori is thought to increase CRC risk by inducing gut microbiota alterations. It is known that H. pylori infection not only impacts the gastric microbiota at the site of infection but also leads to changes in bacterial colonization in the distal large intestine. Considering that the gut microbiome plays a driving role in CRC, H. pylori infection emerges as a key factor responsible for promoting changes in microbiome signatures that could contribute to tumor development. Within this review, we want to focus on the interplay between H. pylori infection, changes in the intestinal microbiota, and intestinal immunity. In addition, the effects of H. pylori antibiotic eradication therapy will be discussed.
Journal Article
Helicobacter pylori promotes colorectal carcinogenesis by deregulating intestinal immunity and inducing a mucus-degrading microbiota signature
Objective Helicobacter pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbour a nearly twofold increased risk to develop colorectal cancer (CRC). However, a direct causal and functional connection between H. pylori infection and colon cancer is lacking.DesignWe infected two Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive analysis of H. pylori-induced changes in intestinal immune responses and epithelial signatures via flow cytometry, chip cytometry, immunohistochemistry and single cell RNA sequencing. Microbial signatures were characterised and evaluated in germ-free mice and via stool transfer experiments.Results H. pylori infection accelerated tumour development in Apc-mutant mice. We identified a unique H. pylori-driven immune alteration signature characterised by a reduction in regulatory T cells and pro-inflammatory T cells. Furthermore, in the intestinal and colonic epithelium, H. pylori induced pro-carcinogenic STAT3 signalling and a loss of goblet cells, changes that have been shown to contribute—in combination with pro-inflammatory and mucus degrading microbial signatures—to tumour development. Similar immune and epithelial alterations were found in human colon biopsies from H. pylori-infected patients. Housing of Apc-mutant mice under germ-free conditions ameliorated, and early antibiotic eradication of H. pylori infection normalised the tumour incidence to the level of uninfected controls.ConclusionsOur studies provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis. Therefore, implementation of H. pylori status into preventive measures of CRC should be considered.
Journal Article
Mitochondrial function controls intestinal epithelial stemness and proliferation
Control of intestinal epithelial stemness is crucial for tissue homeostasis. Disturbances in epithelial function are implicated in inflammatory and neoplastic diseases of the gastrointestinal tract. Here we report that mitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis. Using intestinal epithelial cell (IEC)-specific mouse models, we show that loss of HSP60, a mitochondrial chaperone, activates the mitochondrial unfolded protein response (MT-UPR) and results in mitochondrial dysfunction. HSP60-deficient crypts display loss of stemness and cell proliferation, accompanied by epithelial release of WNT10A and RSPO1. Sporadic failure of Cre-mediated
Hsp60
deletion gives rise to hyperproliferative crypt foci originating from OLFM4
+
stem cells. These effects are independent of the MT-UPR-associated transcription factor CHOP. In conclusion, compensatory hyperproliferation of HSP60
+
escaper stem cells suggests paracrine release of WNT-related factors from HSP60-deficient, functionally impaired IEC to be pivotal in the control of the proliferative capacity of the stem cell niche.
It is unclear what role mitochondrial function plays in maintaining intestinal epithelial cell (IEC) homeostasis. Here, the authors deplete a mitochondrial chaperone, heat shock protein 60 (HSP60) in IEC and observe a loss of stemness and cell proliferation, and suggest this is accompanied by a compensatory release of WNT-related factors.
Journal Article
Effects of Helicobacter pylori adhesin HopQ binding to CEACAM receptors in the human stomach
Helicobacter pylori has developed several strategies using its diverse virulence factors to trigger and, at the same time, limit the host’s inflammatory responses in order to establish a chronic infection in the human stomach. One of the virulence factors that has recently received more attention is a member of the Helicobacter outer membrane protein family, the adhesin HopQ, which binds to the human Carcinoembryonic Antigen-related Cell Adhesion Molecules (CEACAMs) on the host cell surface. The HopQ-CEACAM interaction facilitates the translocation of the cytotoxin-associated gene A (CagA), an important effector protein of H. pylori , into host cells via the Type IV secretion system (T4SS). Both the T4SS itself and CagA are important virulence factors that are linked to many aberrant host signaling cascades. In the last few years, many studies have emphasized the prerequisite role of the HopQ-CEACAM interaction not only for the adhesion of this pathogen to host cells but also for the regulation of cellular processes. This review summarizes recent findings about the structural characteristics of the HopQ-CEACAM complex and the consequences of this interaction in gastric epithelial cells as well as immune cells. Given that the upregulation of CEACAMs is associated with many H. pylori -induced gastric diseases including gastritis and gastric cancer, these data may enable us to better understand the mechanisms of H. pylori ’s pathogenicity.
Journal Article
Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance
Infection with the gastric bacterial pathogen Helicobacter pylori is typically contracted in early childhood and often persists for decades. The immunomodulatory properties of H. pylori that allow it to colonize humans persistently are believed to also account for H. pylori ’s protective effects against allergic and chronic inflammatory diseases. H. pylori infection efficiently reprograms dendritic cells (DCs) toward a tolerogenic phenotype and induces regulatory T cells (Tregs) with highly suppressive activity in models of allergen-induced asthma. We show here that two H. pylori virulence determinants, the γ-glutamyl transpeptidase GGT and the vacuolating cytotoxin VacA, contribute critically and nonredundantly to H. pylori ’s tolerizing effects on murine DCs in vitro and in vivo. The tolerance-promoting effects of both factors are independent of their described suppressive activity on T cells. Isogenic H. pylori mutants lacking either GGT or VacA are incapable of preventing LPS-induced DC maturation and fail to drive DC tolerization as assessed by induction of Treg properties in cocultured naive T cells. The Δ ggt and Δ vacA mutants colonize mice at significantly reduced levels, induce stronger T-helper 1 (Th1) and T-helper 17 (Th17) responses, and/or trigger more severe gastric pathology. Both factors promote the efficient induction of Tregs in vivo, and VacA is required to prevent allergen-induced asthma. The defects of the Δ ggt mutant in vitro and in vivo are phenocopied by pharmacological inhibition of the transpeptidase activity of GGT in all readouts. In conclusion, our results reveal the molecular players and mechanistic basis for H. pylori -induced immunomodulation, promoting persistent infection and conferring protection against allergic asthma.
Journal Article
Dynamics of spike-and nucleocapsid specific immunity during long-term follow-up and vaccination of SARS-CoV-2 convalescents
Anti-viral immunity continuously declines over time after SARS-CoV-2 infection. Here, we characterize the dynamics of anti-viral immunity during long-term follow-up and after BNT162b2 mRNA-vaccination in convalescents after asymptomatic or mild SARS-CoV-2 infection. Virus-specific and virus-neutralizing antibody titers rapidly declined in convalescents over 9 months after infection, whereas virus-specific cytokine-producing polyfunctional T cells persisted, among which IL-2-producing T cells correlated with virus-neutralizing antibody titers. Among convalescents, 5% of individuals failed to mount long-lasting immunity after infection and showed a delayed response to vaccination compared to 1% of naïve vaccinees, but successfully responded to prime/boost vaccination. During the follow-up period, 8% of convalescents showed a selective increase in virus-neutralizing antibody titers without accompanying increased frequencies of circulating SARS-CoV-2-specific T cells. The same convalescents, however, responded to vaccination with simultaneous increase in antibody and T cell immunity revealing the strength of mRNA-vaccination to increase virus-specific immunity in convalescents.
Waning immunity to SARS-CoV-2 is of concern. Here the authors follow spike- and nucleocapsid specific immunity in convalescent individuals for 9 months observing a decline in antibody levels but persisting T cell response. Vaccination approximately 11 months after infection boosts antibody and T cell immunity.
Journal Article
Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double-strand breaks and a DNA damage response in its host cells
The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors—p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)—and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.
Journal Article