A gut-derived metabolite alters brain activity and anxiety behaviour in mice.

Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary components are chemically transformed by the microbiota1 and gut-derived metabolites are disseminated to all organs, including the brain2. In mice, the gut microbiota impacts behaviour3, modulates neurotransmitter production in the gut and brain4,5, and influences brain development and myelination patterns6,7. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment8. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes7,9-14. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.

Remote control of mechanochemical reactions under physiological conditions using biocompatible focused ultrasound.

External control of chemical reactions in biological settings with spatial and temporal precision is a grand challenge for noninvasive diagnostic and therapeutic applications. While light is a conventional stimulus for remote chemical activation, its penetration is severely attenuated in tissues, which limits biological applicability. On the other hand, ultrasound is a biocompatible remote energy source that is highly penetrant and offers a wide range of functional tunability. Coupling ultrasound to the activation of specific chemical reactions under physiological conditions, however, remains a challenge. Here, we describe a synergistic platform that couples the selective mechanochemical activation of mechanophore-functionalized polymers with biocompatible focused ultrasound (FUS) by leveraging pressure-sensitive gas vesicles (GVs) as acousto-mechanical transducers. The power of this approach is illustrated through the mechanically triggered release of covalently bound fluorogenic and therapeutic cargo molecules from polymers containing a masked 2-furylcarbinol mechanophore. Molecular release occurs selectively in the presence of GVs upon exposure to FUS under physiological conditions. These results showcase the viability of this system for enabling remote control of specific mechanochemical reactions with spatiotemporal precision in biologically relevant settings and demonstrate the translational potential of polymer mechanochemistry.

Magneto-acoustic protein nanostructures for non-invasive imaging of tissue mechanics in vivo.

Measuring cellular and tissue mechanics inside intact living organisms is essential for interrogating the roles of force in physiological and disease processes. Current agents for studying the mechanobiology of intact, living organisms are limited by poor light penetration and material stability. Magnetomotive ultrasound is an emerging modality for real-time in vivo imaging of tissue mechanics. Nonetheless, it has poor sensitivity and spatiotemporal resolution. Here we describe magneto-gas vesicles (MGVs), protein nanostructures based on gas vesicles and magnetic nanoparticles that produce differential ultrasound signals in response to varying mechanical properties of surrounding tissues. These hybrid nanomaterials significantly improve signal strength and detection sensitivity. Furthermore, MGVs enable non-invasive, long-term and quantitative measurements of mechanical properties within three-dimensional tissues and in vivo fibrosis models. Using MGVs as novel contrast agents, we demonstrate their potential for non-invasive imaging of tissue elasticity, offering insights into mechanobiology and its application to disease diagnosis and treatment.

Ultrasensitive ultrasound imaging of gene expression with signal unmixing.

Acoustic reporter genes (ARGs) that encode air-filled gas vesicles enable ultrasound-based imaging of gene expression in genetically modified bacteria and mammalian cells, facilitating the study of cellular function in deep tissues. Despite the promise of this technology for biological research and potential clinical applications, the sensitivity with which ARG-expressing cells can be visualized is currently limited. Here we present burst ultrasound reconstructed with signal templates (BURST)-an ARG imaging paradigm that improves the cellular detection limit by more than 1,000-fold compared to conventional methods. BURST takes advantage of the unique temporal signal pattern produced by gas vesicles as they collapse under acoustic pressure above a threshold defined by the ARG. By extracting the unique pattern of this signal from total scattering, BURST boosts the sensitivity of ultrasound to image ARG-expressing cells, as demonstrated in vitro and in vivo in the mouse gastrointestinal tract and liver. Furthermore, in dilute cell suspensions, BURST imaging enables the detection of gene expression in individual bacteria and mammalian cells. The resulting abilities of BURST expand the potential use of ultrasound for non-invasive imaging of cellular functions.

Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures.

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' subcellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs' primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon reaction with the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. Importantly, this was achieved by replacing only a fraction of GvpA with the tagged version. We used fluorescence images of the tagged GVs to study the GV size and distance distributions within these cells. This bioorthogonal and fractional labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts.

The mammalian microbiome has many important roles in health and disease, and genetic engineering is enabling the development of microbial therapeutics and diagnostics. A key determinant of the activity of both natural and engineered microorganisms in vivo is their location within the host organism. However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance owing to light scattering or require radioactive tracers. Here we introduce acoustic reporter genes, which are genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available inexpensive technique with deep tissue penetration and high spatial resolution. These constructs are based on gas vesicles, a unique class of gas-filled protein nanostructures that are expressed primarily in water-dwelling photosynthetic organisms as a means to regulate buoyancy. Heterologous expression of engineered gene clusters encoding gas vesicles allows Escherichia coli and Salmonella typhimurium to be imaged noninvasively at volumetric densities below 0.01% with a resolution of less than 100 µm. We demonstrate the imaging of engineered cells in vivo in proof-of-concept models of gastrointestinal and tumour localization, and develop acoustically distinct reporters that enable multiplexed imaging of cellular populations. This technology equips microbial cells with a means to be visualized deep inside mammalian hosts, facilitating the study of the mammalian microbiome and the development of diagnostic and therapeutic cellular agents.

Gas Vesicle-Blood Interactions Enhance Ultrasound Imaging Contrast.

Gas vesicles (GVs) are genetically encoded, air-filled protein nanostructures of broad interest for biomedical research and clinical applications, acting as imaging and therapeutic agents for ultrasound, magnetic resonance, and optical techniques. However, the biomedical applications of GVs as systemically injectable nanomaterials have been hindered by a lack of understanding of GVs' interactions with blood components, which can significantly impact in vivo behavior. Here, we investigate the dynamics of GVs in the bloodstream using a combination of ultrasound and optical imaging, surface functionalization, flow cytometry, and mass spectrometry. We find that erythrocytes and serum proteins bind to GVs and shape their acoustic response, circulation time, and immunogenicity. We show that by modifying the GV surface we can alter these interactions and thereby modify GVs' in vivo performance. These results provide critical insights for the development of GVs as agents for nanomedicine.

Geometric effects in gas vesicle buckling under ultrasound.

Acoustic reporter genes based on gas vesicles (GVs) have enabled the use of ultrasound to noninvasively visualize cellular function in vivo. The specific detection of GV signals relative to background acoustic scattering in tissues is facilitated by nonlinear ultrasound imaging techniques taking advantage of the sonomechanical buckling of GVs. However, the effect of geometry on the buckling behavior of GVs under exposure to ultrasound has not been studied. To understand such geometric effects, we developed computational models of GVs of various lengths and diameters and used finite element simulations to predict their threshold buckling pressures and postbuckling deformations. We demonstrated that the GV diameter has an inverse cubic relation to the threshold buckling pressure, whereas length has no substantial effect. To complement these simulations, we experimentally probed the effect of geometry on the mechanical properties of GVs and the corresponding nonlinear ultrasound signals. The results of these experiments corroborate our computational predictions. This study provides fundamental insights into how geometry affects the sonomechanical properties of GVs, which, in turn, can inform further engineering of these nanostructures for high-contrast, nonlinear ultrasound imaging.

Genetically encodable materials for non-invasive biological imaging.

Many questions in basic biology and medicine require the ability to visualize the function of specific cells and molecules inside living organisms. In this context, technologies such as ultrasound, optoacoustics and magnetic resonance provide non-invasive imaging access to deep-tissue regions, as used in many laboratories and clinics to visualize anatomy and physiology. In addition, recent work has enabled these technologies to image the location and function of specific cells and molecules inside the body by coupling the physics of sound waves, nuclear spins and light absorption to unique protein-based materials. These materials, which include air-filled gas vesicles, capsid-like nanocompartments, pigment-producing enzymes and transmembrane transporters, enable new forms of biomolecular and cellular contrast. The ability of these protein-based contrast agents to be genetically encoded and produced by cells creates opportunities for unprecedented in vivo studies of cellular function, while their amenability to genetic engineering enables atomic-level design of their physical, chemical and biological properties.

Acoustic biosensors for ultrasound imaging of enzyme activity.

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors-molecules that 'light up' in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce nonlinear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract.

Publisher Correction: Acoustic biosensors for ultrasound imaging of enzyme activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Self-assembly of protein superstructures by physical interactions under cytoplasm-like conditions.

The structure-driven assembly of multimeric protein complexes and the formation of intracellular phase-like protein condensates have been the subject of intense research. However, the assembly of larger superstructures comprising cellular components, such as protein nanoparticles driven by general physical rather than specific biochemical interactions, remains relatively uncharacterized. Here, we use gas vesicles (GVs)-genetically encoded protein nanoparticles that form ordered intracellular clusters-as a model system to study the forces driving multiparticle assembly under cytoplasm-like conditions. Our calculations and experimental results show that the ordered assembly of GVs can be achieved by screening their mutual electrostatic repulsion with electrolytes and creating a crowding force with dissolved macromolecules. The precise balance of these forces results in different packing configurations. Biomacromolecules such as polylysine and DNA are capable of driving GV clustering. These results provide basic insights into how physically driven interactions affect the formation of protein superstructures, offer guidance for manipulating nanoparticle assembly in cellular environments through synthetic biology methods, and inform research on the biotechnology applications of GVs.

Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy.

Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure, and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, expanding the capabilities of this powerful technology for three-dimensional biological imaging.

Nanoscale Heat Transfer from Magnetic Nanoparticles and Ferritin in an Alternating Magnetic Field.

Recent suggestions of nanoscale heat confinement on the surface of synthetic and biogenic magnetic nanoparticles during heating by radio frequency-alternating magnetic fields have generated intense interest because of the potential utility of this phenomenon for noninvasive control of biomolecular and cellular function. However, such confinement would represent a significant departure from the classical heat transfer theory. Here, we report an experimental investigation of nanoscale heat confinement on the surface of several types of iron oxide nanoparticles commonly used in biological research, using an all-optical method devoid of the potential artifacts present in previous studies. By simultaneously measuring the fluorescence of distinct thermochromic dyes attached to the particle surface or dissolved in the surrounding fluid during radio frequency magnetic stimulation, we found no measurable difference between the nanoparticle surface temperature and that of the surrounding fluid for three distinct nanoparticle types. Furthermore, the metalloprotein ferritin produced no temperature increase on the protein surface nor in the surrounding fluid. Experiments mimicking the designs of previous studies revealed potential sources of the artifacts. These findings inform the use of magnetic nanoparticle hyperthermia in engineered cellular and molecular systems.

Acoustic biomolecules enhance hemodynamic functional ultrasound imaging of neural activity.

Hemodynamic functional ultrasound imaging (fUS) of neural activity provides a unique combination of spatial coverage, spatiotemporal resolution and compatibility with freely moving animals. However, deep and transcranial monitoring of brain activity and the imaging of dynamics in slow-flowing blood vessels remains challenging. To enhance fUS capabilities, we introduce biomolecular hemodynamic enhancers based on gas vesicles (GVs), genetically encodable ultrasound contrast agents derived from buoyant photosynthetic microorganisms. We show that intravenously infused GVs enhance ultrafast Doppler ultrasound contrast and visually-evoked hemodynamic contrast in transcranial fUS of the mouse brain. This hemodynamic contrast enhancement is smoother than that provided by conventional microbubbles, allowing GVs to more reliably amplify neuroimaging signals.

Achieving Spatial and Molecular Specificity with Ultrasound-Targeted Biomolecular Nanotherapeutics.

The precise targeting of cells in deep tissues is one of the primary goals of nanomedicine. However, targeting a specific cellular population within an entire organism is challenging due to off-target effects and the need for deep tissue delivery. Focused ultrasound can reduce off-targeted effects by spatially restricting the delivery or action of molecular constructs to specific anatomical sites. Ultrasound can also increase the efficiency of nanotherapeutic delivery into deep tissues by enhancing the permeability of tissue boundaries, promoting convection, or depositing energy to actuate cellular activity. In this review we focus on the interface between biomolecular engineering and focused ultrasound and describe the applications of this intersection in neuroscience, oncology, and synthetic biology. Ultrasound can be used to trigger the transport of therapeutic payloads into a range of tissues, including specific regions of the brain, where it can be targeted with millimeter precision through intact skull. Locally delivered molecular constructs can then control specific cells and molecular pathways within the targeted region. When combined with viral vectors and engineered neural receptors, this technique enables noninvasive control of specific circuits and behaviors. The penetrant energy of ultrasound can also be used to more directly actuate micro- and nanotherapeutic constructs, including microbubbles, vaporizable nanodroplets, and polymeric nanocups, which nucleate cavitation upon ultrasound exposure, leading to local mechanical effects. In addition, it was recently discovered that a unique class of acoustic biomolecules-genetically encodable nanoscale protein structures called gas vesicles-can be acoustically "detonated" as sources of inertial cavitation. This enables the targeted disruption of selected cells within the area of insonation by gas vesicles that are engineered to bind cell surface receptors. It also facilitates ultrasound-triggered release of molecular payloads from engineered therapeutic cells heterologously expressing intracellular gas vesicles. Finally, focused ultrasound energy can be used to locally elevate tissue temperature and activate temperature-sensitive proteins and pathways. The elevation of temperature allows noninvasive control of gene expression in vivo in cells engineered to express thermal bioswitches. Overall, the intersection of biomolecular engineering, nanomaterials and focused ultrasound can provide unparalleled specificity in controlling, modulating, and treating physiological processes in deep tissues.

Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures.

Non-invasive biological imaging requires materials capable of interacting with deeply penetrant forms of energy such as magnetic fields and sound waves. Here, we show that gas vesicles (GVs), a unique class of gas-filled protein nanostructures with differential magnetic susceptibility relative to water, can produce robust contrast in magnetic resonance imaging (MRI) at sub-nanomolar concentrations, and that this contrast can be inactivated with ultrasound in situ to enable background-free imaging. We demonstrate this capability in vitro, in cells expressing these nanostructures as genetically encoded reporters, and in three model in vivo scenarios. Genetic variants of GVs, differing in their magnetic or mechanical phenotypes, allow multiplexed imaging using parametric MRI and differential acoustic sensitivity. Additionally, clustering-induced changes in MRI contrast enable the design of dynamic molecular sensors. By coupling the complementary physics of MRI and ultrasound, this nanomaterial gives rise to a distinct modality for molecular imaging with unique advantages and capabilities.

Tunable thermal bioswitches for in vivo control of microbial therapeutics.

Temperature is a unique input signal that could be used by engineered microbial therapeutics to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, we present two families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32-46 °C. We integrate these molecular bioswitches into thermal logic circuits and demonstrate their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications.

In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ.

Directed evolution continues to expand the capabilities of complex biomolecules for a range of applications, such as adeno-associated virus vectors for gene therapy; however, advances in library design and selection strategies are key to develop variants that overcome barriers to clinical translation. To address this need, we applied structure-guided SCHEMA recombination of the multimeric adeno-associated virus (AAV) capsid to generate a highly diversified chimeric library with minimal structural disruption. A stringent in vivo Cre-dependent selection strategy was implemented to identify variants that transduce adult neural stem cells (NSCs) in the subventricular zone. A novel variant, SCH9, infected 60% of NSCs and mediated 24-fold higher GFP expression and a 12-fold greater transduction volume than AAV9. SCH9 utilizes both galactose and heparan sulfate as cell surface receptors and exhibits increased resistance to neutralizing antibodies. These results establish the SCHEMA library as a valuable tool for directed evolution and SCH9 as an effective gene delivery vector to investigate subventricular NSCs.

Ultraparamagnetic Cells Formed through Intracellular Oxidation and Chelation of Paramagnetic Iron.

Making cells magnetic is a long-standing goal of chemical biology, aiming to enable the separation of cells from complex biological samples and their visualization in vivo using magnetic resonance imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have been largely unsuccessful due to the stringent required chemical conditions. Here, we introduce an alternative approach to making cells magnetic, focused on biochemically maximizing cellular paramagnetism. We show that a novel genetic construct combining the functions of ferroxidation and iron chelation enables engineered bacterial cells to accumulate iron in "ultraparamagnetic" macromolecular complexes, allowing these cells to be trapped with magnetic fields and imaged with MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, nuclear magnetic resonance, biochemical assays, and computational modeling to elucidate the unique mechanisms and capabilities of this paramagnetic concept.