A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation.

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

Nucleation of the destruction complex on the centrosome accelerates degradation of ß-catenin and regulates Wnt signal transmission.

Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate ß-catenin stability. Overexpressed DC scaffolds undergo liquid-liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in ß-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of ß-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3ß partitioning on the centrosome controls ß-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle.

Design, Synthesis, and Application of a Water-soluble Photocage for Aqueous Cyclopentadiene-based Diels-Alder Photoclick Chemistry in Hydrogels.

Spatiotemporally functionalized hydrogels have exciting applications in tissue engineering, but their preparation often relies on radical-based strategies that can be deleterious in biological settings. Herein, the computationally guided design, synthesis, and application of a water-soluble cyclopentadienone-norbornadiene (CPD-NBD) adduct is disclosed as a diene photocage for radical-free Diels-Alder photopatterning. We show that this scalable CPD-NBD derivative is readily incorporated into hydrogel formulations, providing gels that can be patterned with dienophiles upon 365 nm uncaging of cyclopentadiene. Patterning is first visualized through conjugation of cyanine dyes, then biological utility is highlighted by patterning peptides to direct cellular adhesion. Finally, the ease of use and versatility of this CPD-NBD derivative is demonstrated by direct incorporation into a commercial 3D printing resin to enable the photopatterning of structurally complex, printed hydrogels.

Triggered Functional Dynamics of AsLOV2 by Time-Resolved Electron Paramagnetic Resonance at High Magnetic Fields.

We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein To Facilitate the Detection of SARS-CoV-2.

Management of the coronavirus disease 2019 (COVID-19) pandemic requires widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among them RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here, we propose an alternative method we call PEARL (precipitation-enhanced analyte retrieval) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers performance comparable to that of commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material.

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.

Light-based control of metabolic flux through assembly of synthetic organelles.

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink.

While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal-bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA's cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed.

An explainable map of human gastruloid morphospace reveals gastrulation failure modes and predicts teratogens.

Human gastrulation is a critical stage of development where many pregnancies fail due to poorly understood mechanisms. Using the 2D gastruloid, a stem cell model of human gastrulation, we combined high-throughput drug perturbations and mathematical modelling to create an explainable map of gastruloid morphospace. This map outlines patterning outcomes in response to diverse perturbations and identifies variations in canonical patterning and failure modes. We modeled morphogen dynamics to embed simulated gastruloids into experimentally-determined morphospace to explain how developmental parameters drive patterning. Our model predicted and validated the two greatest sources of patterning variance: cell density-based modulations in Wnt signaling and SOX2 stability. Assigning these parameters as axes of morphospace imparted interpretability. To demonstrate its utility, we predicted novel teratogens that we validated in zebrafish. Overall, we show how stem cell models of development can be used to build a comprehensive and interpretable understanding of the set of developmental outcomes.

Field-domain rapid-scan EPR at 240GHz for studies of protein functional dynamics at room temperature.

We present field-domain rapid-scan (RS) electron paramagnetic resonance (EPR) at 8.6T and 240GHz. To enable this technique, we upgraded a home-built EPR spectrometer with an FPGA-enabled digitizer and real-time processing software. The software leverages the Hilbert transform to recover the in-phase (I) and quadrature (Q) channels, and therefore the raw absorptive and dispersive signals, χ' and χ'', from their combined magnitude (I2+Q2). Averaging a magnitude is simpler than real-time coherent averaging and has the added benefit of permitting long-timescale signal averaging (up to at least 2.5×106 scans) because it eliminates the effects of source-receiver phase drift. Our rapid-scan (RS) EPR provides a signal-to-noise ratio that is approximately twice that of continuous wave (CW) EPR under the same experimental conditions, after scaling by the square root of acquisition time. We apply our RS EPR as an extension of the recently reported time-resolved Gd-Gd EPR (TiGGER) [Maity et al., 2023], which is able to monitor inter-residue distance changes during the photocycle of a photoresponsive protein through changes in the Gd-Gd dipolar couplings. RS, opposed to CW, returns field-swept spectra as a function of time with 10ms time resolution, and thus, adds a second dimension to the static field transients recorded by TiGGER. We were able to use RS TiGGER to track time-dependent and temperature-dependent kinetics of AsLOV2, a light-activated phototropin domain found in oats. The results presented here combine the benefits of RS EPR with the improved spectral resolution and sensitivity of Gd chelates at high magnetic fields. In the future, field-domain RS EPR at high magnetic fields may enable studies of other real-time kinetic processes with time resolutions that are otherwise difficult to access in the solution state.

Prior Fc receptor activation primes macrophages for increased sensitivity to IgG via long-term and short-term mechanisms.

Macrophages measure the "eat-me" signal immunoglobulin G (IgG) to identify targets for phagocytosis. We tested whether prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc receptor. To temporally control Fc receptor activation, we engineered an Fc receptor that is activated by the light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that subthreshold Fc receptor activation primes mouse bone-marrow-derived macrophages to be more sensitive to IgG in future encounters. Macrophages that have previously experienced subthreshold Fc receptor activation eat more IgG-bound human cancer cells. Increased phagocytosis occurs by two discrete mechanisms-a short- and long-term priming. Long-term priming requires new protein synthesis and Erk activity. Short-term priming does not require new protein synthesis and correlates with an increase in Fc receptor mobility. Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.

YAP dysregulation triggers hypertrophy by CCN2 secretion and TGFß uptake in human pluripotent stem cell-derived cardiomyocytes.

Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in b-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet, how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway, specifically its effector molecule YAP, has been demonstrated to be reactivated in pathological hypertrophic growth. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, YAP, is altered 1) by changes in the biomechanics of HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates to confirm the hypercontractile phenotype in MYH7 mutants. We next modulate contractility in healthy and disease hiPSC-CMs by treatment with positive and negative inotropic drugs and demonstrate a correlative relationship between contractility and YAP activity. We further demonstrate the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation. We conclude that the overactivation of YAP, possibly initiated and driven by hypercontractility, correlates with excessive CCN2 secretion (connective tissue growth factor), enhancing cardiac fibroblast/myofibroblast transition and production of known hypertrophic signaling molecule TGFß. Our study suggests YAP being an indirect player in the initiation of hypertrophic growth and fibrosis in HCM. Our results provide new insights into HCM progression and bring forth a testbed for therapeutic options in treating HCM.

Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape.

The integrated stress response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered a photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics, we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR, which demonstrated the dependence of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. A record of this paper's transparent peer review process is included in the supplemental information.

Prior Fc Receptor activation primes macrophages for increased sensitivity to IgG via long term and short term mechanisms.

Macrophages measure the 'eat-me' signal IgG to identify targets for phagocytosis. We wondered if prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc Receptor. To temporally control Fc Receptor activation, we engineered an Fc Receptor that is activated by light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that Fc Receptor activation primes macrophages to be more sensitive to IgG in future encounters. Macrophages that have previously experienced Fc Receptor activation eat more IgG-bound cancer cells. Increased phagocytosis occurs by two discrete mechanisms - a short- and long-term priming. Long term priming requires new protein synthesis and Erk activity. Short term priming does not require new protein synthesis and correlates with an increase in Fc Receptor mobility. Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.

Diels-Alder Photoclick Patterning of Extracellular Matrix for Spatially Controlled Cell Behaviors.

Strategies that mimic the spatial complexity of natural tissues can provide cellular scaffolds to probe fundamental questions in cell biology and offer new materials for regenerative medicine. Here, the authors demonstrate a light-guided patterning platform that uses natural engineered extracellular matrix (ECM) proteins as a substrate to program cellular behaviors. A photocaged diene which undergoes Diels-Alder-based click chemistry upon uncaging with 365 nm light is utilized. By interfacing with commercially available maleimide dienophiles, patterning of common ECM proteins (collagen, fibronectin Matrigel, laminin) with readily purchased functional small molecules and growth factors is achieved. Finally, the use of this platform to spatially control ERK activity and migration in mammalian cells is highlighted, demonstrating programmable cell behavior through patterned chemical modification of natural ECM.

Extracellular Optogenetics at the Interface of Synthetic Biology and Materials Science.

We review fundamental mechanisms and applications of OptoGels: hydrogels with light-programmable properties endowed by photoswitchable proteins ("optoproteins") found in nature. Light, as the primary source of energy on earth, has driven evolution to develop highly-tuned functionalities, such as phototropism and circadian entrainment. These functions are mediated through a growing family of optoproteins that respond to the entire visible spectrum ranging from ultraviolet to infrared by changing their structure to transmit signals inside of cells. In a recent series of articles, engineers and biochemists have incorporated optoproteins into a variety of extracellular systems, endowing them with photocontrollability. While other routes exist for dynamically controlling material properties, light-sensitive proteins have several distinct advantages, including precise spatiotemporal control, reversibility, substrate selectivity, as well as biodegradability and biocompatibility. Available conjugation chemistries endow OptoGels with a combinatorially large design space determined by the set of optoproteins and polymer networks. These combinations result in a variety of tunable material properties. Despite their potential, relatively little of the OptoGel design space has been explored. Here, we aim to summarize innovations in this emerging field and highlight potential future applications of these next generation materials. OptoGels show great promise in applications ranging from mechanobiology, to 3D cell and organoid engineering, and programmable cell eluting materials.

Algal p-coumaric acid induces oxidative stress and siderophore biosynthesis in the bacterial symbiont Phaeobacter inhibens.

The marine alpha-proteobacterium Phaeobacter inhibens engages in intermittent symbioses with microalgae. The symbiosis is biphasic and concludes in a parasitic phase, during which the bacteria release algaecidal metabolites in response to algal p-coumaric acid (pCA). The cell-wide effects of pCA on P. inhibens remain unknown. Herein, we report a microarray-based transcriptomic study and find that genes related to the oxidative stress response and secondary metabolism are upregulated most, while those associated with energy production and motility are downregulated in the presence of pCA. Among genes upregulated is a previously unannotated biosynthetic gene cluster and, using a combination of gene deletions and metabolic profiling, we show that it gives rise to an unreported siderophore, roseobactin. The simultaneous production of algaecides and roseobactin in the parasitic phase allows the bacteria to take up any iron that is released from dying algal cells, thereby securing a limited micronutrient.