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The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air–liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1β, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.

Abstract The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-β, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-β signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

IntroductionCervical cancer represents one of the main causes of female, cancer-related mortality worldwide. The majority of cancers are caused by human papillomaviruses such as HPV16 and HPV18. As chemotherapeutic resistance to first-line platinum treatment is still a predominant clinical challenge in advanced cervical cancer, novel treatment options including combinatorial therapies are urgently required to overcome chemotherapeutic resistance. Inhibition of A Disintegrin And Metalloproteinase (ADAM)-family members, heavily involved in tumour progression of a vast range of solid tumours, strongly improved response to chemotherapeutic treatment in other tumour entities including ovarian cancer.MethodsWe established two- and three-dimensional models derived from three traditional cervical cancer cell lines and ectocervical cancer-derived organoids. Following characterisation, these models were used to investigate their response to cisplatin treatment in the absence and presence of ADAM inhibitors using viability assays and automated live cell imaging.ResultsThe pivotal role of the metalloprotease ADAM17 driving chemotherapy resistance was detectable in all ectocervical cultures irrespective of the model system used, whereas ADAM10 inhibition was predominantly effective only in loosely aggregated spheroids. We showed prominent differences regarding treatment responses between 2D monolayers compared to 3D spheroid and 3D organoid model systems. Particularly, the organoid system, regarded as the closest representation of primary tumours, exhibited reliably the combinatorial effect of ADAM17 inhibition and cisplatin in all three individual donors.DiscussionAs two- and three-dimensional models of the same cell lines differ in their responses to chemotherapy it is essential to validate treatment strategies in more advanced model systems representing the patient situation more realistically. Ectocervical organoids showed reliable results regarding treatment responses closely mimicking the primary tumours and could therefore serve as an important tool for personalized medicine in cervical cancer. These findings strengthen the role of ADAM17 as a potential novel target for combinatorial treatments to overcome chemoresistance in cervical cancer.

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.

Abstract Background High-content screening (HCS) experiments generate complex data from multiple object features for each cell within a treated population. Usually, these data are analyzed by using population-averaged values of the features of interest, increasing the amount of false positives and the need for intensive follow-up validation. Therefore, there is a strong need for novel approaches with reproducible hit prediction by identifying significantly altered cell populations. Results Here we describe SOPRA, a workflow for analyzing image-based HCS data based on regression analysis of non-averaged object features from cell populations, which can be run on hundreds of samples using different cell features. Following plate-wise normalization, the values are counted within predetermined binning intervals, generating unique frequency distribution profiles (histograms) for each population, which are then normalized to control populations (control-based normalization). These control-normalized frequency distribution profiles are analyzed using the Bioconductor R-package maSigPro, originally developed to analyze time profiles. However, statistically significant altered frequency distributions are also identified by maSigPro when integrating it into the SOPRA workflow. Finally, significantly changed profiles can be used to generate a heatmap from which altered cell populations with similar phenotypes can be identified, enabling the detection of siRNAs and compounds with the same ‘on-target’ profile and reducing the number of false positive hits. Conclusions SOPRA is a novel analysis workflow for the detection of statistically significant normalized frequency distribution profiles of cellular features generated in high-throughput RNAi screens. For the validation of the SOPRA software workflow, a screen for cell cycle progression was used. We were able to identify such profiles for siRNA-mediated gene perturbations and chemical inhibitors of different cell cycle stages. The SOPRA software is freely available from Github.

Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here the authors identify a BMP feedback loop between the stomach epithelium and surrounding stroma that controls gland homeostasis and demonstrate its interruption upon infection with H. pylori.

Here, Koster et al., model human papillomavirus and Chlamydia coinfection dynamics in patient-derived ectocervical organoids, and characterize the effects of multiple infections in the cellular microenvironment, potentially contributing to neoplasia.

Viruses manipulate host cell pathways to support infection. Here the authors show that SARS-CoV-2 infection modulates cellular metabolism and limits autophagy, and identify druggable host pathways for virus inhibition.

Colibactin-producing pks+ Escherichia coli are frequent constituents of the human intestinal microbiota. Here, the authors show that short exposure of cells to pks+ E. coli induces chromosomal aberrations, genomic instability, and multiple features of transformation reminiscent of colorectal cancer.

The bacterial pathogen Helicobacter pylori is known for its ability to induce DNA double-strand breaks in the genome of its target cells. Here, we show that H. pylori-induced DNA damage and replication stress occurs in S-phase cells as a result of R-loop-mediated transcription/replication conflicts that are triggered by activation of the ALPK1/TIFA/NF-κB signaling axis.

Epithelial turnover in the colon requires stem cells in the crypt that express the R-spondin receptor Lgr5. Here, the authors show that regeneration after colon injury involving loss of Lgr5+ and Axin2+ cells requires stromal derived Rspo3-dependent reprogramming of Lgr4+ differentiated cells, including Krt20+ enterocytes.

Chronic infections of the fallopian tubes with Chlamydia trachomatis can cause scarring and infertility. Here, the authors show that the pathogen alters stem cell differentiation and DNA methylation in human fallopian tube organoids, suggesting a potential link to cellular ageing and malignant transformation.

Abstract Background Deregulated c-Abl activity has been intensively studied in a variety of solid tumors and leukemia. The class-I carcinogen Helicobacter pylori (Hp) activates the non-receptor tyrosine kinase c-Abl to phosphorylate the oncoprotein cytotoxin-associated gene A (CagA). The role of c-Abl in CagA-dependent pathways is well established; however, the knowledge of CagA-independent c-Abl processes is scarce. Methods c-Abl phosphorylation and localization were analyzed by immunostaining and immunofluorescence. Interaction partners were identified by tandem-affinity purification. Cell elongation and migration were analyzed in transwell-filter experiments. Apoptosis and cell survival were examined by FACS analyses and MTT assays. In mice experiments and human biopsies, the involvement of c-Abl in Hp pathogenesis was investigated. Results Here, we investigated the activity and subcellular localization of c-Abl in vitro and in vivo and unraveled the contribution of c-Abl in CagA-dependent and -independent pathways to gastric Hp pathogenesis. We report a novel mechanism and identified strong c-Abl threonine 735 phosphorylation (pAblT735) mediated by the type-IV secretion system (T4SS) effector D-glycero-β-D-manno-heptose-1,7-bisphosphate (βHBP) and protein kinase C (PKC) as a new c-Abl kinase. pAblT735 interacted with 14–3-3 proteins, which caused cytoplasmic retention of c-Abl, where it potentiated Hp-mediated cell elongation and migration. Further, the nuclear exclusion of pAblT735 attenuated caspase-8 and caspase-9-dependent apoptosis. Importantly, in human patients suffering from Hp-mediated gastritis c-Abl expression and pAblT735 phosphorylation were drastically enhanced as compared to type C gastritis patients or healthy individuals. Pharmacological inhibition using the selective c-Abl kinase inhibitor Gleevec confirmed that c-Abl plays an important role in Hp pathogenesis in a murine in vivo model. Conclusions In this study, we identified a novel regulatory mechanism in Hp-infected gastric epithelial cells by which Hp determines the subcellular localization of activated c-Abl to control Hp-mediated EMT-like processes while decreasing cell death.

Summary: Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.

ABSTRACT Carcinoma of the gallbladder (GBC) is the most frequent tumor of the biliary tract. Despite epidemiological studies showing a correlation between chronic infection with Salmonella enterica Typhi/Paratyphi A and GBC, the underlying molecular mechanisms of this fatal connection are still uncertain. The murine serovar Salmonella Typhimurium has been shown to promote transformation of genetically predisposed cells by driving mitogenic signaling. However, insights from this strain remain limited as it lacks the typhoid toxin produced by the human serovars Typhi and Paratyphi A. In particular, the CdtB subunit of the typhoid toxin directly induces DNA breaks in host cells, likely promoting transformation. To assess the underlying principles of transformation, we used gallbladder organoids as an infection model for Salmonella Paratyphi A. In this model, bacteria can invade epithelial cells, and we observed host cell DNA damage. The induction of DNA double-strand breaks after infection depended on the typhoid toxin CdtB subunit and extended to neighboring, non-infected cells. By cultivating the organoid derived cells into polarized monolayers in air-liquid interphase, we could extend the duration of the infection, and we observed an initial arrest of the cell cycle that does not depend on the typhoid toxin. Non-infected intoxicated cells instead continued to proliferate despite the DNA damage. Our study highlights the importance of the typhoid toxin in causing genomic instability and corroborates the epidemiological link between Salmonella infection and GBC. IMPORTANCE Bacterial infections are increasingly being recognized as risk factors for the development of adenocarcinomas. The strong epidemiological evidence linking Helicobacter pylori infection to stomach cancer has paved the way to the demonstration that bacterial infections cause DNA damage in the host cells, initiating transformation. In this regard, the role of bacterial genotoxins has become more relevant. Salmonella enterica serovars Typhi and Paratyphi A have been clinically associated with gallbladder cancer. By harnessing the stem cell potential of cells from healthy human gallbladder explant, we regenerated and propagated the epithelium of this organ in vitro and used these cultures to model S. Paratyphi A infection. This study demonstrates the importance of the typhoid toxin, encoded only by these specific serovars, in causing genomic instability in healthy gallbladder cells, posing intoxicated cells at risk of malignant transformation.

ABSTRACT Multiple studies have demonstrated rapid bacterial genome evolution during chronic infection with Helicobacter pylori. In contrast, little was known about genetic changes during the first stages of infection, when selective pressure is likely to be highest. Using single-molecule, real-time (SMRT) and Illumina sequencing technologies, we analyzed genome and methylome evolution during the first 10 weeks of infection by comparing the cag pathogenicity island (cagPAI)-negative H. pylori challenge strain BCS 100 with pairs of H. pylori reisolates from gastric antrum and corpus biopsy specimens of 10 human volunteers who had been infected with this strain as part of a vaccine trial. Most genetic changes detected in the reisolates affected genes with a surface-related role or a predicted function in peptide uptake. Apart from phenotypic changes of the bacterial envelope, a duplication of the catalase gene was observed in one reisolate, which resulted in higher catalase activity and improved survival under oxidative stress conditions. The methylomes also varied in some of the reisolates, mostly by activity switching of phase-variable methyltransferase (MTase) genes. The observed in vivo mutation spectrum was remarkable for a very high proportion of nonsynonymous mutations. Although the data showed substantial within-strain genome diversity in the challenge strain, most antrum and corpus reisolates from the same volunteers were highly similar to each other, indicating that the challenge infection represents a major selective bottleneck shaping the transmitted population. Our findings suggest rapid in vivo selection of H. pylori during early-phase infection providing adaptation to different individuals by common mechanisms of genetic and epigenetic alterations. IMPORTANCE Exceptional genetic diversity and variability are hallmarks of Helicobacter pylori, but the biological role of this plasticity remains incompletely understood. Here, we had the rare opportunity to investigate the molecular evolution during the first weeks of H. pylori infection by comparing the genomes and epigenomes of H. pylori strain BCS 100 used to challenge human volunteers in a vaccine trial with those of bacteria reisolated from the volunteers 10 weeks after the challenge. The data provide molecular insights into the process of establishment of this highly versatile pathogen in 10 different human individual hosts, showing, for example, selection for changes in host-interaction molecules as well as changes in epigenetic methylation patterns. The data provide important clues to the early adaptation of H. pylori to new host niches after transmission, which we believe is vital to understand its success as a chronic pathogen and develop more efficient treatments and vaccines.

Summary: Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of GTPases and conveys host defense against intracellular bacteria and parasites. During infection, hGBP1 is recruited to pathogen-containing vacuoles, such as Chlamydia trachomatis inclusions, restricts pathogenic growth, and induces the activation of the inflammasome pathway. hGBP1 has a unique catalytic activity to hydrolyze guanosine triphosphate (GTP) to guanosine monophosphate (GMP) in two consecutive cleavage steps. However, the functional significance of this activity in host defense remains elusive. Here, we generate a structure-guided mutant that specifically abrogates GMP production, while maintaining fast cooperative GTP hydrolysis. Complementation experiments in human monocytes/macrophages show that hGBP1-mediated GMP production is dispensable for restricting Chlamydia trachomatis growth but is necessary for inflammasome activation. Mechanistically, GMP is catabolized to uric acid, which in turn activates the NLRP3 inflammasome. Our study demonstrates that the unique enzymology of hGBP1 coordinates bacterial growth restriction and inflammasome signaling.

An endogenous molecular clockwork drives various cellular pathways including metabolism and the cell cycle. Its dysregulation is able to prompt pathological phenotypes including cancer. Besides dramatic metabolic alterations, cancer cells display severe changes in the clock phenotype with likely consequences in tumor progression and treatment response. In this study, we use a comprehensive systems-driven approach to investigate the effect of clock disruption on metabolic pathways and its impact on drug response in a cellular model of colon cancer progression. We identified distinctive time-related transcriptomic and metabolic features of a primary tumor and its metastatic counterpart. A mapping of the expression data to a comprehensive genome-scale reconstruction of human metabolism allowed for the in-depth functional characterization of 24 h-oscillating transcripts and pointed to a clock-driven metabolic reprogramming in tumorigenesis. In particular, we identified a set of five clock–regulated glycolysis genes, ALDH3A2, ALDOC, HKDC1, PCK2, and PDHB with differential temporal expression patterns. These findings were validated in organoids and in primary fibroblasts isolated from normal colon and colon adenocarcinoma from the same patient. We further identified a reciprocal connection of HKDC1 to the clock in the primary tumor, which is lost in the metastatic cells. Interestingly, a disruption of the core-clock gene BMAL1 impacts on HKDC1 and leads to a time-dependent rewiring of metabolism, namely an increase in glycolytic activity, as well as changes in treatment response. This work provides novel evidence regarding the complex interplay between the circadian clock and metabolic alterations in carcinogenesis and identifies new connections between both systems with pivotal roles in cancer progression and response to therapy. Keywords: Circadian clock, Colorectal cancer, Metabolic rewiring, Tumor progression, High-throughput circadian data, Metabolic network reconstruction, Circadian regulation of metabolism, Treatment response, Glycolysis

As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease. Keywords: Distal airway epithelial cells, Influenza A virus, Alveolar epithelial cells, Lung disease

Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1β- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen’s T4SS.

The intracellular human bacterial pathogen Chlamydia trachomatis pursues effective strategies to protect infected cells against death-inducing stimuli. Here, we show that Chlamydia trachomatis infection evokes 3-phosphoinositide-dependent protein kinase-1 (PDPK1) signaling to ensure the completion of its developmental cycle, further leading to the phosphorylation and stabilization of MYC. Using biochemical approaches and imaging we demonstrate that Chlamydia-induced PDPK1-MYC signaling induces host hexokinase II (HKII), which becomes enriched and translocated to the mitochondria. Strikingly, preventing the HKII interaction with mitochondria using exogenous peptides triggers apoptosis of infected cells as does inhibiting either PDPK1 or MYC, which also disrupts intracellular development of Chlamydia trachomatis. These findings identify a previously unknown pathway activated by Chlamydia infection, which exhibits pro-carcinogenic features. Targeting the PDPK1-MYC-HKII-axis may provide a strategy to overcome therapeutic resistance of infection.

Summary: Chlamydia trachomatis (Ctr) causes a range of infectious diseases and is epidemiologically associated with cervical and ovarian cancers. To obtain a panoramic view of Ctr-induced signaling, we performed global phosphoproteomic and transcriptomic analyses. We identified numerous Ctr phosphoproteins and Ctr-regulated host phosphoproteins. Bioinformatics analysis revealed that these proteins were predominantly related to transcription regulation, cellular growth, proliferation, and cytoskeleton organization. In silico kinase substrate motif analysis revealed that MAPK and CDK were the most overrepresented upstream kinases for upregulated phosphosites. Several of the regulated host phosphoproteins were transcription factors, including ETS1 and ERF, that are downstream targets of MAPK. Functional analysis of phosphoproteome and transcriptome data confirmed their involvement in epithelial-to-mesenchymal transition (EMT), a phenotype that was validated in infected cells, along with the essential role of ERK1/2, ETS1, and ERF for Ctr replication. Our data reveal the extent of Ctr-induced signaling and provide insights into its pro-carcinogenic potential. : Zadora et al. performed an integrated global phosphoproteomic and transcriptomic analysis, revealing a comprehensive map of Chlamydia-induced host cell signaling and identifying transcription factors ETS1 and ERF, which drive epithelial-to-mesenchymal transition. These insights provide mechanistic clues to Chlamydia pathogenesis and serve as an important resource for future studies. Keywords: Chlamydia trachomatis, signaling, human papillomavirus, transcription factors, cervical cancer, ovarian cancer, human primary cells

Chikungunya virus is a mosquito transmitted untreatable emergent pathogen that causes joint pain and fever. Here the authors perform a host genome-wide loss-of-function screen to identify targets for chikungunya antiviral drugs and validate hits using a mouse model of chikungunya infection.

ABSTRACT Cervical and ovarian cancers exhibit characteristic mutational signatures that are reminiscent of mutational processes, including defective homologous recombination (HR) repair. How these mutational processes are initiated during carcinogenesis is largely unclear. Chlamydia trachomatis infections are epidemiologically associated with cervical and ovarian cancers. Previously, we showed that C. trachomatis induces DNA double-strand breaks (DSBs) but suppresses Ataxia-telangiectasia mutated (ATM) activation and cell cycle checkpoints. The mechanisms by which ATM regulation is modulated and its consequences for the repair pathway in C. trachomatis-infected cells remain unknown. Here, we found that Chlamydia bacteria interfere with the usual response of PP2A to DSBs. As a result, PP2A activity remains high, as the level of inhibitory phosphorylation at Y307 remains unchanged following C. trachomatis-induced DSBs. Protein-protein interaction analysis revealed that C. trachomatis facilitates persistent interactions of PP2A with ATM, thus suppressing ATM activation. This correlated with a remarkable lack of homologous recombination (HR) repair in C. trachomatis-infected cells. Chemical inhibition of PP2A activity in infected cells released ATM from PP2A, resulting in ATM phosphorylation. Activated ATM was then recruited to DSBs and initiated downstream signaling, including phosphorylation of MRE11 and NBS1 and checkpoint kinase 2 (Chk2)-mediated activation of the G2/M cell cycle checkpoint in C. trachomatis-infected cells. Further, PP2A inhibition led to the restoration of C. trachomatis-suppressed HR DNA repair function. Taking the data together, this study revealed that C. trachomatis modulates PP2A signaling to suppress ATM activation to prevent cell cycle arrest, thus contributing to a deficient high-fidelity HR pathway and a conducive environment for mutagenesis. IMPORTANCE Chlamydia trachomatis induces DNA double-strand breaks in host cells but simultaneously inhibits proper DNA damage response and repair mechanisms. This may render host cells prone to loss of genetic integrity and transformation. Here we show that C. trachomatis prevents activation of the key DNA damage response mediator ATM by preventing the release from PP2A, leading to a complete absence of homologous recombination repair in host cells.

Infection with the human pathogen Helicobacter pylori (H. pylori) is a major risk factor for gastric cancer. Since the bacterium exerts multiple genotoxic effects, we examined the circumstances of DNA damage accumulation and identified regions within the host genome with high susceptibility to H. pylori-induced damage. Infection impaired several DNA repair factors, the extent of which depends on a functional cagPAI. This leads to accumulation of a unique DNA damage pattern, preferentially in transcribed regions and proximal to telomeres, in both gastric cell lines and primary gastric epithelial cells. The observed pattern correlates with focal amplifications in adenocarcinomas of the stomach and partly overlaps with known cancer genes. We thus demonstrate an impact of a bacterial infection directed toward specific host genomic regions and describe underlying characteristics that make such regions more likely to acquire heritable changes during infection, which could contribute to cellular transformation.