Biology subjects

Englert, F.

Publications and source records attributed to Englert, F..

6 recordsLinked to original sources

bioRxiv2026

Targeted DNA nicking enables efficient, single-step and counterselection-free editing of bacteriophage genomes

Genetic manipulation of bacteriophages is essential for interrogating phage biology and advancing antimicrobial therapies. However, current genome editing approaches can be inefficient, require multiple steps, or drastically reduce phage titers. Here, we show that targeted DNA nicking enables template-mediated editing of phage genomes in one step without reducing phage titers. Using T7 phage, we show that Cas9-mediated nicking achieved up to 100% recombination across multiple loci, including substitutions and deletions of up to 200 bp and insertions of up to 500 bp, all while preserving phage titers. Editing in T7 was RecA-independent and extended to other phages. Leveraging high titers, we engineered a T7 library of over 440,000 tail-fiber mutants, with isolated mutants restoring infection of two LPS-deficient Escherichia coli hosts by shifting recognition to core LPS components. Overall, DNA nicking is a simple and distinct editing strategy that can advance phage genome engineering, genetic interrogation, and antimicrobial development.

synthetic biology↗

bioRxiv2026

A type II Thoeris anti-phage defense enacts growth arrest independent of NAD depletion

Thoeris immune systems protect bacteria against invading phages through production of a signaling molecule by ThsB that activates the effector ThsA. Across the four defined types, the two most dominant (I and II) have been associated with abortive infection, with type I acting through the depletion of the essential coenzyme NAD+. Here, we show that a previously uncharacterized type II Thoeris system from Escherichia coli ATCC 25922 deviates from this paradigm. This system consists of the effector protein ThsA and two distinct signaling proteins ThsB1 and ThsB2. Heterologous expression of thsA and thsB2 confers anti-phage defense, while thsB1 is cytotoxic when expressed in common E. coli lab strains. Furthermore, while phage infection drives growth arrest, we could not detect any measurable decrease in NAD+ levels as well as standard markers of cell death. Together, these results suggest that Thoeris contains even greater functional diversity within the defined system types.

microbiology↗

bioRxiv2023

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans

Type IV-A CRISPR-Cas systems are primarily encoded on plasmids and form multi-subunit ribonucleoprotein complexes with unknown biological functions. In contrast to other CRISPR-Cas types, they lack the archetypical CRISPR acquisition module and encode a DinG helicase instead of a nuclease component. Type IV-A3 systems are carried by large conjugative plasmids that often harbor multiple antibiotic-resistance genes. Although their CRISPR array contents suggest a role in inter-plasmid conflicts, this function and the underlying mechanisms have remained unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 CRISPR-Cas system co-opts the type I-E adaptation machinery from its clinical Klebsiella pneumoniae host to update its CRISPR array. Furthermore, we demonstrate that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By targeting plasmid core functions, type IV-A3 can prevent the uptake of incoming plasmids, limit their horizontal transfer, and destabilize co-residing plasmids, altogether supporting type IV-A3s involvement in plasmid competition. Collectively, our findings shed light on the molecular mechanisms and ecological function of type IV-A3 systems and have broad implications for understanding and countering the spread of antibiotic resistance in clinically relevant strains.

microbiology↗

bioRxiv2022

Chemical screening of food-related chemicals for human fatty liver risk: Combining high content imaging of cellular responses with in vitro to in vivo extrapolation

1Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent human disease with accumulating evidence linking its pathophysiology and co-morbidities to chemical exposures. The complex pathophysiology of NAFLD has limited the elucidation of potential chemical etiologies. In this study we generated a high-content imaging analysis method for the simultaneous quantification of sentinel steatosis cellular markers in chemically exposed human liver cells in vitro combined with a computational model for the extrapolation of human oral equivalent doses (OED). First, the in vitro test method was generated using 14 reference chemicals with known capacities to induce cellular alterations in nuclear morphology, lipid accumulation, mitochondrial membrane potential and oxidative stress. These effects were quantified on a single cell- and population-level, and then, using physiologically based pharmacokinetic modelling and reverse dosimetry, OEDs were extrapolated from these in vitro data. The extrapolated OEDs were confirmed to be within biologically relevant exposure ranges for the reference chemicals. Next, we tested 14 chemicals found in food, selected from thousands of putative chemicals on the basis of structure-based prediction for nuclear receptor activation. Amongst these, orotic acid had an extrapolated OED overlapping with realistic exposure ranges. By the strategy developed in this study, we were able to characterize known NAFLD-inducing chemicals and translate to data scarce food-related chemicals, amongst which we identified orotic acid to induce steatosis. This strategy addresses needs of next generation risk assessment, and can be used as a first chemical prioritization hazard screening step in a tiered approach to identify chemical risk factors for NAFLD.

pharmacology and toxicology↗

bioRxiv2021

Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons

CRISPR-Cas biology and technologies have been largely shaped to-date by the characterization and use of single-effector nucleases. In contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons. HIGHLIGHTS[bullet] PAM-DETECT for rapid determination of PAMs for Type I CRISPR-Cas systems in TXTL [bullet]Mining of Type I orthologs and characterization of extensively self-targeting systems [bullet]TXTL-based assessment of DNA target recognition and transposition by CRISPR transposons [bullet]Identification of a distinct branch of Type I-B CRISPR transposons

molecular biology↗

bioRxiv2021

Functional metabolic phenotyping of human pancreatic ductal adenocarcinoma

Pancreatic Ductal Adenocarcinoma (PDAC) lacks targeted treatment options. Although subtypes with transcriptome-based distinct lineage and differentiation features have been identified, deduced clinically actionable targets remain elusive. We here investigate functional metabolic features of the classical and QM (quasi-mesenchymal)/basal-like PDAC subtypes potentially exploitable for non-invasive subtype differentiation and therapeutic intervention. A collection of human PDAC cell lines, primary patient derived cells (PDC), patient derived xenografts (PDX) and patient PDAC samples were transcriptionally stratified into the classical and QM subtype. Functional metabolic analyses including targeted and non-targeted metabolite profiling (matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)), seahorse metabolic flux assays and metabolic drug targeting were performed. Hyperpolarized 13C-magnetic resonance spectroscopy (HP-MRS) of PDAC xenografts was used for in vivo detection of intra-tumoral [1-13C]pyruvate and [1-13C]lactate metabolism. We identified glycolysis and lipid metabolism/fatty acid oxidation as transcriptionally preserved metabolic pathways in QM and classical PDAC subtype respectively. However, these metabolic cues were not unambiguously functionally linked to one subtype. Striking functional metabolic heterogeneity was observed especially in primary patient derived cells with only individual samples representing high dependence on glycolysis or mitochondrial oxidation. Of note, QM cells actively use the glycolytic product lactate as oxidative mitochondrial fuel. Using HP-MRS, we were able to non-invasively differentiate glycolytic tumor xenografts with high intratumoral [1-13C]pyruvate to [1-13C]lactate conversion in vivo. Although PDAC transcriptomes indicate molecular subtype-associated distinct metabolic pathways, we found substantial functional metabolic heterogeneity independent of the molecular subtype. Non-invasive identification of highly glycolytic tumors by [1-13C]pyruvate/lactate HP-MRS support individualized metabolic targeting approaches.

cancer biology↗