Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer.

Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free survival in advanced-stage HGSOC. Phospho- and interaction proteomics tied CT45 to DNA damage pathways through direct interaction with the PP4 phosphatase complex. In vitro, CT45 regulated PP4 activity, and its high expression led to increased DNA damage and platinum sensitivity. CT45-derived HLA class I peptides, identified by immunopeptidomics, activate patient-derived cytotoxic T cells and promote tumor cell killing. This study highlights the power of clinical cancer proteomics to identify targets for chemo- and immunotherapy and illuminate their biological roles.

Heat shock induces premature transcript termination and reconfigures the human transcriptome.

The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.

Cistromic Reprogramming of the Diurnal Glucocorticoid Hormone Response by High-Fat Diet.

The glucocorticoid receptor (GR) is a potent metabolic regulator and a major drug target. While GR is known to play integral roles in circadian biology, its rhythmic genomic actions have never been characterized. Here we mapped GR's chromatin occupancy in mouse livers throughout the day and night cycle. We show how GR partitions metabolic processes by time-dependent target gene regulation and controls circulating glucose and triglycerides differentially during feeding and fasting. Highlighting the dominant role GR plays in synchronizing circadian amplitudes, we find that the majority of oscillating genes are bound by and depend on GR. This rhythmic pattern is altered by high-fat diet in a ligand-independent manner. We find that the remodeling of oscillatory gene expression and postprandial GR binding results from a concomitant increase of STAT5 co-occupancy in obese mice. Altogether, our findings highlight GR's fundamental role in the rhythmic orchestration of hepatic metabolism.

ADAMTS-7 Modulates Atherosclerotic Plaque Formation by Degradation of TIMP-1.

BACKGROUND: The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis. METHODS: Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. RESULTS: In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site. CONCLUSIONS: ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events.

Comprehensive chromatin proteomics resolves functional phases of pluripotency and identifies changes in regulatory components.

The establishment of cellular identity is driven by transcriptional and epigenetic regulators of the chromatin proteome - the chromatome. Comprehensive analyses of the chromatome composition and dynamics can therefore greatly improve our understanding of gene regulatory mechanisms. Here, we developed an accurate mass spectrometry (MS)-based proteomic method called Chromatin Aggregation Capture (ChAC) followed by Data-Independent Acquisition (DIA) and analyzed chromatome reorganizations during major phases of pluripotency. This enabled us to generate a comprehensive atlas of proteomes, chromatomes, and chromatin affinities for the ground, formative and primed pluripotency states, and to pinpoint the specific binding and rearrangement of regulatory components. These comprehensive datasets combined with extensive analyses identified phase-specific factors like QSER1 and JADE1/2/3 and provide a detailed foundation for an in-depth understanding of mechanisms that govern the phased progression of pluripotency. The technical advances reported here can be readily applied to other models in development and disease.

EPOP Functionally Links Elongin and Polycomb in Pluripotent Stem Cells.

The cellular plasticity of pluripotent stem cells is thought to be sustained by genomic regions that display both active and repressive chromatin properties. These regions exhibit low levels of gene expression, yet the mechanisms controlling these levels remain unknown. Here, we describe Elongin BC as a binding factor at the promoters of bivalent sites. Biochemical and genome-wide analyses show that Elongin BC is associated with Polycomb Repressive Complex 2 (PRC2) in pluripotent stem cells. Elongin BC is recruited to chromatin by the PRC2-associated factor EPOP (Elongin BC and Polycomb Repressive Complex 2 Associated Protein, also termed C17orf96, esPRC2p48, E130012A19Rik), a protein expressed in the inner cell mass of the mouse blastocyst. Both EPOP and Elongin BC are required to maintain low levels of expression at PRC2 genomic targets. Our results indicate that keeping the balance between activating and repressive cues is a more general feature of chromatin in pluripotent stem cells than previously appreciated.

TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation.

DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.

Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.

Phosphorylation of the HP1ß hinge region sequesters KAP1 in heterochromatin and promotes the exit from naïve pluripotency.

Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.

Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism.

BACKGROUND: Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. METHODS: Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. RESULTS: The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. CONCLUSIONS: The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target.

Anti-inflammatory functions of the glucocorticoid receptor require DNA binding.

The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.

A proteomic atlas of the neointima identifies novel druggable targets for preventive therapy.

AIMS: In-stent restenosis is a complication after coronary stenting associated with morbidity and mortality. Here, we sought to investigate the molecular processes underlying neointima formation and to identify new treatment and prevention targets. METHODS AND RESULTS: Neointima formation was induced by wire injury in mouse femoral arteries. High-accuracy proteomic measurement of single femoral arteries to a depth of about 5000 proteins revealed massive proteome remodelling, with more than half of all proteins exhibiting expression differences between injured and non-injured vessels. We observed major changes in the composition of the extracellular matrix and cell migration processes. Among the latter, we identified the classical transient receptor potential channel 6 (TRPC6) to drive neointima formation. While Trpc6-/- mice presented reduced neointima formation compared to wild-type mice (1.44 ± 0.39 vs. 2.16 ± 0.48, P = 0.01), activating or repressing TRPC6 in human vascular smooth muscle cells resulted in increased [vehicle 156.9 ± 15.8 vs. 1-oleoyl-2-acetyl-sn-glycerol 179.1 ± 8.07 (103 pixels), P = 0.01] or decreased migratory capacity [vehicle 130.0 ± 26.1 vs. SAR7334 111.4 ± 38.0 (103 pixels), P = 0.04], respectively. In a cohort of individuals with angiographic follow-up (n = 3068, males: 69.9%, age: 59 ± 11 years, follow-up 217.1 ± 156.4 days), homozygous carriers of a common genetic variant associated with elevated TRPC6 expression were at increased risk of restenosis after coronary stenting (adjusted odds ratio 1.49, 95% confidence interval 1.08-2.05; P = 0.01). CONCLUSIONS: Our study provides a proteomic atlas of the healthy and injured arterial wall that can be used to define novel factors for therapeutic targeting. We present TRPC6 as an actionable target to prevent neointima formation secondary to vascular injury and stent implantation.

Kreuth V initiative: European consensus proposals for treatment of hemophilia using standard products, extended half-life coagulation factor concentrates and non-replacement therapies.

This report contains the updated consensus recommendations for optimal hemophilia care produced in 2019 by three Working Groups (WG) on behalf of the European Directorate for Quality of Medicines and Healthcare in the frame of the Kreuth V Initiative. WG1 recommended access to prophylaxis for all patients, the achievement of plasma factor trough levels of at least 3-5% when extended half-life factor VIII (FVIII) and FIX products are used, a personalized treatment regimen, and a choice of chromogenic assays for treatment monitoring. It was also emphasized that innovative therapies should be supervised by hemophilia comprehensive care centers. WG2 recommended mandatory collection of postmarketing data to assure the long-term safety and efficacy of new hemophilia therapies, the establishment of national patient registries including the core data recommended by the European Medicines Agency and the International Society on Thrombosis and Haemostasis, with adequate support under public control, and greater collaboration to facilitate a comprehensive data evaluation throughout Europe. WG3 discussed methodological aspects of hemophilia care in the context of access decisions, particularly for innovative therapies, and recommended that clinical studies should be designed to provide the quality of evidence needed by regulatory authorities, HTA bodies and healthcare providers. The dialogue between all stakeholders in hemophilia care and patient organizations should be fostered to implement these recommendations.

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression.

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function.

Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes.

High-resolution mass spectrometry (MS)-based proteomics is a powerful method for the identification of soluble protein complexes and large-scale affinity purification screens can decode entire protein interaction networks. In contrast, protein complexes residing on chromatin have been much more challenging, because they are difficult to purify and often of very low abundance. However, this is changing due to recent methodological and technological advances in proteomics. Proteins interacting with chromatin marks can directly be identified by pulldowns with synthesized histone tails containing posttranslational modifications (PTMs). Similarly, pulldowns with DNA baits harbouring single nucleotide polymorphisms or DNA modifications reveal the impact of those DNA alterations on the recruitment of transcription factors. Accurate quantitation - either isotope-based or label free - unambiguously pinpoints proteins that are significantly enriched over control pulldowns. In addition, protocols that combine classical chromatin immunoprecipitation (ChIP) methods with mass spectrometry (ChIP-MS) target gene regulatory complexes in their in-vivo context. Similar to classical ChIP, cells are crosslinked with formaldehyde and chromatin sheared by sonication or nuclease digested. ChIP-MS baits can be proteins in tagged or endogenous form, histone PTMs, or lncRNAs. Locus-specific ChIP-MS methods would allow direct purification of a single genomic locus and the proteins associated with it. There, loci can be targeted either by artificial DNA-binding sites and corresponding binding proteins or via proteins with sequence specificity such as TAL or nuclease deficient Cas9 in combination with a specific guide RNA. We predict that advances in MS technology will soon make such approaches generally applicable tools in epigenetics.

Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the ß1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the ß1 subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants.

Reprogramming neuroblastoma by diet-enhanced polyamine depletion.

Neuroblastoma is a highly lethal childhood tumor derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fueled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, with the inhibitor difluoromethylornithine showing clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumor differentiation, and profound survival gains in the TH-MYCN mouse model. Specifically, an arginine/proline-free diet decreases the polyamine precursor ornithine and augments tumor polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at adenosine-ending codons. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by the diet-drug combination, favors a pro-differentiation proteome. These results suggest that the genes of specific cellular programs have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of pediatric cancers.

Independent control of COVID-19 vaccines by EU Official Control Authority Batch Release: challenges, strengths and successes.

Vaccines have been a key tool in stemming the tide of the COVID-19 pandemic. The rapid development of effective vaccines against COVID-19, together with their regulatory approval and wide scale distribution has been achieved in an impressively short period thanks to the intense efforts of many. In parallel to vaccine development, the EU considered it important to prepare for the independent control of the COVID-19 vaccines, including testing, to help ensure that only vaccines that comply with the approved quality requirements reach the public and to help improve/increase public confidence in the vaccines. The existing EU Official Control Authority Batch Release (OCABR) system, co-ordinated by the European Directorate for the Quality of Medicines and HealthCare (EDQM), was able to effectively respond to the need, through rapid co-ordination, work-sharing, advance planning and early interaction with manufacturers, the Coalition for Epidemic Preparedness Innovation (CEPI) and regulatory authorities. The Official Medicines Control Laboratories (OMCLs) involved in the OCABR activity, using the strength of the established system in the OCABR network and adaptations to the crisis conditions, were ready to release the first COVID-19 vaccine batches, after protocol review and testing, at the time of the conditional marketing authorisation for each of the COVID-19 vaccines, with no delay for batches reaching the public. Thanks to the dedication of resources by the EU and national authorities as well as by the EDQM, this was done without impacting the release of the other vaccines and human blood and plasma derived medicinal products, essential for public health. Transparency and communication of practices were important factors to support reliance on the OCABR outcome in non-EU countries, with the goal to improve access to vaccines in Europe and beyond. An overview of the process, legal background, challenges and successes of OCABR for COVID-19 vaccines as well as a look at the international perspective and lessons learned is provided.

Clostridium scindens secretome suppresses virulence gene expression of Clostridioides difficile in a bile acid-independent manner.

Clostridioides difficile infection (CDI) is a major health concern and one of the leading causes of hospital-acquired diarrhea in many countries. C. difficile infection is challenging to treat as C. difficile is resistant to multiple antibiotics. Alternative solutions are needed as conventional treatment with broad-spectrum antibiotics often leads to recurrent CDI. Recent studies have shown that specific microbiota-based therapeutics such as bile acids (BAs) are promising approaches to treat CDI. Clostridium scindens encodes the bile acid-induced (bai) operon that carries out 7-alpha-dehydroxylation of liver-derived primary BAs to secondary BAs. This biotransformation is thought to increase the antibacterial effects of BAs on C. difficile. Here, we used an automated multistage fermentor to study the antibacterial actions of C. scindens and BAs on C. difficile in the presence/absence of a gut microbial community derived from healthy human donor fecal microbiota. We observed that C. scindens inhibited C. difficile growth when the medium was supplemented with primary BAs. Transcriptomic analysis indicated upregulation of C. scindens bai operon and suppressed expression of C. difficile exotoxins that mediate CDI. We also observed BA-independent antibacterial activity of the secretome from C. scindens cultured overnight in a medium without supplementary primary BAs, which suppressed growth and exotoxin expression in C. difficile mono-culture. Further investigation of the molecular basis of our observation could lead to a more specific treatment for CDI than current approaches. IMPORTANCE There is an urgent need for new approaches to replace the available treatment options against Clostridioides difficile infection (CDI). Our novel work reports a bile acid-independent reduction of C. difficile growth and virulence gene expression by the secretome of Clostridium scindens. This potential treatment combined with other antimicrobial strategies could facilitate the development of alternative therapies in anticipation of CDI and in turn reduce the risk of antimicrobial resistance.