Geoffrey Chang - US grants

Abstract Not Provided.

structural biology; transport proteins; crystallization; drug /agent; biomedical resource;

structural biology; transport proteins; protein structure; crystallization; multidrug resistance; biomedical resource;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Multi-drug resistance is a very significant health problem that has both medical and pharmacological concerns. The cause of the mdr phenotype is the result of a net decrease in the concentration of drug levels within the cell. This reduction occurs by two fundamental mechanisms that either lowers the membrane permeability, which leads to a decreased rate of drug entry across the lipid bilayer (Class I), or directly increasing the rate of drug removal from the cell (Class II). Both of these mechanisms are accomplished via a series of energy-dependent energy efflux pumps. Recently, we have obtained 3D crystals of a second distinct class of mdr transporters (Class II). The goal of this proposal is to determine the atomic structure of these mdr transporters. Hence, we will be able to discover the structural basis of mdr for this family of integral membrane proteins. We propose to screen and collect data from our membrane protein crystals and solve the structure of mdr transporter at SSRL.

X ray crystallography; bioimaging /biomedical imaging

DESCRIPTION (provided by applicant): The liver and kidney excrete from the body a wide array of positively charged organic molecules of physiological, pharmacological and toxicological significance. Carrier-mediated secretion of these organic cations (OCs), particularly by the kidney, has a profound influence on the pharmacokinetics of these compounds and, importantly, OC secretion is the site of many clinically significant drug-drug interactions. The active and rate-limiting step in OC secretion involves the carrier-mediated exit of accumulated OCs across the luminal membrane of renal and hepatic epithelia. The molecular identify of this process involves the newly identified Multidrug And Toxin Extrusion proteins, MATE1 and MATE2-K. Although now clearly understood to play a significant role in OC secretion, virtually nothing is known about the molecular determinants of substrate interaction with these transporters. In this revised proposal, we take advantage of the recent solution of the x- ray structure of a prototypic member of the MATE family of transport protein (NorM). We have used the NorM structure to develop a homology model of human MATE1 and, in this proposal, we outline two sets experiments designed to develop a predictive model of drug interaction with MATE transporters. In Aim 1, we take a ligand-based approach to develop 3D-QSAR/pharmacophore models of substrate/inhibitor interaction with MATE1 and MATE2-K. These data will be interpreted in the context of parallel studies on the integrated activity of these transporters in epithelial models of renal secretion (which, in turn, will be interpreted in the context of studies on the differential distribution of these transporters in human kidney). Aim 2 will employ a target-based approach, using site-directed studies to probe the topology and surface accessibility of MATE1, thereby testing predictions arising from our homology model, and establishing a database designed to probe the functional structure of the protein as determined in a parallel effort to solve the x-ray structure of human MATE1. Aim 2 will also study the substrate translocation pathway of MATE1 in studies that apply (i) proteomic methods to identify peptides and amino acid residues that specifically interact with a photoactivatable probe of the OC/H+ exchanger; and (ii) apply computational methods (steered molecular dynamics) to identify amino acid residues that influence substrate translocation. These studies will play a critical role in establishing models that accurately predict and, ideally, preempt unwanted interactions of cationic drugs in both the kidney and liver.

DESCRIPTION (provided by applicant) Marine pollution is of concern for human health through our exposure to contaminated food from the sea. What remains poorly understood is why some chemicals are persistent, accumulating in marine organisms and then in humans, while others are not. Multidrug resistance (MDR) transporters, belonging to the ATP Binding Cassette (ABC) family, are major biological determinants of intracellular chemical accumulation. While they have been implicated as determinants of environmental chemical persistence and used as tools for predicting availability and efficacy of drugs, they have yet to be systematically applied to predicting persistence of pollutants. The investigators' preliminary data indicate striking functional conservation of the major sub-family types (ABCB, ABCC and ABCG) of xenobiotic eliminating transporters between sea urchins and man. This application explores the molecular basis for this conserved substrate selectivity as a first step towards application of transporter biology to prediction of pollutant persistence. In this project, the investigators will over-express, solubilize, and purify sea urchin multidrug efflux transporter proteins, develop assays for their interaction with major marine pollutants and attempt to determine their high-resolution structures. They will measure their interaction with persistent marine pollutants using anisotropy, ATPase and whole cell assays. By comparing structure and functions of sea urchin with those of mammalian transporter proteins, already available through the TransportPDB pipeline, they will identify conserved residues and structural features that are essential for predicting substrate interaction in the poly-specific binding pocket of these ABC transporters Public Health Relevance: Certain molecular transporters found in sea urchins and other marine organisms determine the uptake of persistent pollutants from the sea. The goal of this high-impact project is to provide a detailed molecular framework for understanding the structure and function of these important transporters. This application fulfills an important need to develop biologically-based tools to predict bioaccumulation and toxicity of marine pollutants as relevant to human health and environment.

DESCRIPTION (provided by applicant): About 40% of all prescribed drugs are cationic at physiological pH and the multidrug Organic Cation Transporters, OCT1 and OCT2, are the initial steps in their hepatic and renal secretion, respectively. Their broad selectivity makes both of these processes principal sites for unwanted drug-drug interactions (DDIs). The impact these transporters have on DDIs, as well as the influence they have on the pharmacokinetics of cationic drugs, makes them a focus of efforts to develop models capable of predicting and, ideally, pre-empting unwanted interactions. Moreover, FDA guidance increasingly urge that New Molecular Entities (NMEs) be tested using defined protocols ('Decision Trees') designed to identify potentials DDIs with OCTs, as well as other multidrug transporters, and the results influence key decisions on the clinical and commercial future of these compounds. The value of these efforts (for both modeling and regulatory recommendations) is, however, compromised by a lack of understanding of two critical issues: (1) we cannot predict with sufficient quantitative accuracy the extent to which novel drugs or preclinical molecules will interact with either OCT1 or OCT2; and (2) we have no theoretical (or empirical) basis upon which to predict whether an inhibitor of OCT activity is itself a substrate for transport. The present proposal describes studies that directly address both these issues by confronting two critical flaws in previous efforts to model drug interactions with OCT1 and OCT2, i.e., the failure to acknowledge the influence of 'substrate' on the 'selectivity' profiles upon which current models of drug interactio with polyspecific OCTs have been based; and the absence of accurate information on the structures of these proteins. To fill these gaps in knowledge we will integrate ligand-based and protein- based approaches for development of structure/function relationships for human OCT1 and OCT2. The plan is organized around two major aims. Aim 1 will develop models of drug interaction with OCT1 and OCT2 using newly developed protocols capable of high throughput and high resolution assessment of the mechanistic basis of substrate-specific ligand interaction with these proteins. These protocols include a means to determine if inhibitors of OCT activity are themselves substrates for transport. We will also introduce a way to share the models we develop via web- and mobile device-based applications. Aim 2 will determine the crystal structure of OCT1 and OCT2, thereby clarifying the structural basis of ligand-interaction with OCTS, including the influence of polymorphisms on substrate- and inhibitor-specific activity profiles. In summary, the proposed program will develop robust computational models of ligand interaction with OCTs that, when integrated with knowledge of the crystal structures of OCT transporters, will increase opportunities to identify novel OCT substrates and inhibitors, thereby holding the promise of assisting in drug development as well as predicting and preempting adverse drug interactions.

Marine pollution is of concern for human health through our exposure to contaminated food from the sea. What remains poorly understood is why some chemicals are persistent, accumulating in marine organisms and then in humans, while others are not. Multidrug resistance (MDR) transporters, belonging to the ATP Binding Cassette (ABC) family, are major biological determinants of intracellular chemical accumulation. While ABC transporters have been implicated as determinants of pollutant persistence, and used as tools for predicting availability and efficacy of drugs, they have yet to be systematically applied to understanding why certain pollutants accumulate.

In this project, a research team at the Scripps Institute of Oceanography will work to develop a structural and functional understanding of multidrug (MDR) ATP Binding Cassette (ABC) transporters and their interaction with global marine pollutants. They will clone, express and purify MDR ABC transporters from sea urchins and other aquatic organisms, and determine their molecular structures. They will also test, in vitro, whether global, persistent pollutants are substrates, inhibitors or not bound (i.e. not recognized) by the transporters. They will extend findings from the in vitro assays to the cellular and organismal level using pollutant accumulation, cytotoxicity and fluorescence competition assays with recombinant marine proteins and morpholino antisense oligonucleotide-mediated loss of function assays in sea urchins, a tractable marine model system. In vitro and in vivo results from marine transporters will be assessed in parallel assays with mammalian (mouse and human) transporters to determine which interaction patterns are conserved, potentially leading to transfer of pollutants from marine organisms to man. The team already has in-hand preliminary data that indicate striking functional conservation of the major subfamily types of xenobiotic eliminating transporters in sea urchins and man.

Broader Impacts: Given the potential risk to human health from marine pollution, there is a need to refine our ability to predict accumulation and develop new tools to enable design of safer chemicals. This project is expected to make significant advances toward that goal. The research team will include several postdoctoral researchers and graduate students.

JOINT FUNDING BY NSF AND NIEHS: The original proposal on which this project is based (R01 ES021985-01) was submitted to the National Institutes of Environmental Health Sciences (NIH/NIEHS) in response to Funding Opportunity Announcement RFA-ES-11-013 , "Oceans, Great Lakes and Human Health (R01)", an opportunity jointly sponsored by NSF. This project is cooperatively funded through separate awards from NSF and NIEHS.

DESCRIPTION (provided by applicant): The metabolic activity of the brain is extraordinary, accounting for nearly 20% of the body's energy demand. As such, it is perhaps unsurprising that a deficit in energy metabolism is associated with multiple forms of chronic neurodegenerative disease. In Alzheimer's Disease (AD), for example, the earliest detectable defect in patients is diminished glucose utilization by the brain. Epidemiological data reinforces the link between dysregulated glucose metabolism, as type 2 diabetes is a significant risk factor for development of AD and cognitive impairment. In fact, the two diseases share several noteworthy features, including insulin resistance (the inability of tissues to properly transport and metabolize glucose in response to insulin), oxidative stress, amyloidosis, cognitive dysfunction, and atrophy of neural tissues. This shared pathogenesis has led to clinical trials which re-purpose medications used to treat type 2 diabetes [including intranasal insulin, incretin analogues, and thiazolidinediones (TZDs)] to treat AD and other neurodegenerative diseases. Unfortunately, each of these approaches suffer from the same drawbacks that limit their use in diabetes, including significant side effects from TZDs mediated by transcriptional nuclear receptor PPAR?. We recently reported that TZDs have a previously undiscovered, pleiotropic effect in which they specifically modulate the activity of an important metabolite transporter - the mitochondrial pyruvate carrier (MPC, Divakaruni et al. 2013). The MPC transports pyruvate from the cytoplasm into mitochondria, and as such is a crucial branch point in cellular metabolism. Mild inhibition of the MPC by TZDs can acutely stimulate glucose uptake into human myocytes, and this effect can be reproduced by the specific MPC inhibitor UK5099. We have extended our work into neurodegenerative disease with promising early data. A low concentration of UK5099 also acutely stimulates glucose uptake in primary rat cortical neurons, and 24 h treatment enhances their ability to oxidize alternative metabolic fuels (such as ketone bodies) and withstand excitotoxic stress. We therefore propose a novel strategy for the treatment of AD: identify drug-like compounds that mildly inhibit the MPC to (i) stimulate cellular glucose uptake and (ii) promote the oxidation of alternative fuels. To achieve this goal, we propose the following: Aim 1: Characterize the response of primary neurons and astrocytes to mild MPC inhibition with further studies of glucose uptake, resistance to excitotoxic death, ROS production, and metabolic flexibility using 13C flux analysis. Aim 2: Determine the high-resolution structures of the human MPC protein complex and functionally important mutants as well as with UK5099 to provide a detailed framework to support drug development efforts. Aim 3: Conduct a chemical screen to identify mild MPC inhibitors, using a battery of follow-on assays in neurons and astrocytes to further optimize a potential lead compound. This Multi-PI project on a novel mitochondrial target merges expertise in bioenergetics, drug discovery, and structural biology into a synergistic program for the treatment of neurodegenerative disease.

Learning requires the conversion of transient experiences into long-lasting changes in neural circuitry. Animal behavior triggers changes in gene expression in small populations of neurons and behaviorally induced genes regulate synapses and neuronal morphology. Yet, it is unclear if changes in gene expression are the cause of behavioral plasticity, or the consequence. This project will develop a new genre of fluorescent reporters that enable the visualization and manipulation of endogenous transcription factors in individual neurons, in real time, and within the brain of behaving animals. During the award period, candidate reporters will be made that recognize six different transcription factors. These reporters will have widespread utility for investigating the molecular mechanisms that support learning in vivo and analysis of populations of neurons that are active during a learning paradigm. The development of these reporters includes ongoing training of undergraduate, graduate, and postgraduate scientists. Student training is optimized with guidance from the CREATE STEM Success Initiative on the UCSD campus.

Inducible transcription factors (ITFs) translate signals that last milliseconds or seconds into changes in cellular function that may persist for hours, days, or longer. This project will develop genetically encoded transcription factor reporters (GETFaRs) that are designed to visualize or manipulate an ITF. GETFaRs are based on molecular scaffolds, engineered through a process of synthetic affinity maturation of camelid nanobodies (Nbs) which bind the endogenous ITF. The Nb protein will be fused to a fluorophore or DNA modifying enzyme, allowing users to visualize or manipulate endogenous transcription factors. A degradation signal (degron) will be incorporated into the Nb near the ITF binding site. Consequently, GETFaRs will be constitutively expressed and rapidly degraded in the cytoplasm. When the ITF is expressed, the GETFaR-ITF interaction will mask the degron, stabilizing the complex. The ITF's nuclear localization signal will translocate the complex into the nucleus, resulting in stabilized GETFaRs that accumulate in the nucleus and stoichiometrically reflect ITF expression. Candidate GETFaRs will be validated in vitro using standard biochemical and imaging techniques and in vivo using two photon imaging of neurons in head fixed mice. Optimal GETFaRs will enable research that 1) monitors or manipulates transcriptional states during learning, 2) studies the emergence of ensembles of co-active neurons within a circuit, 3) probes the dynamics of chromatin and nuclear organization, and 4) analyzes the genome of defined populations of neurons responding to complex, natural stimuli.

PI: Geoffrey Chang (University of California-San Diego)

Co-PIs: Erin Connolly (University of South Carolina), Leon Kochian and Miguel Piñeros (USDA-ARS/Cornell University), Michael Stowell (University of Colorado-Boulder), Julian Schroeder and Milton Saier (University of California, San Diego)

Plants need to take up nutrients from the soil and transport these nutrients to specialized cells throughout the whole plant. These nutrients are transported across plant cell membranes and organelle membranes inside of each plant cell. Transporters are proteins that mediate the passage of virtually every molecule and ion across plant cell membranes. Many natural variants of transport proteins have been shown to be important for key agronomic traits associated with plant growth and yield, and tolerance to abiotic and biotic stresses. However, the fundamental biochemical, functional, and structural characteristics of these proteins are largely unknown. To address these questions and the glaring need for new tools in this research area, we are establishing a new center called the Center for Research on Plant TransporterS (CROPS), devoted to the functional and structural characterization of plant transporters prioritized by their agronomic importance. This center will leverage an existing advanced research infrastructure to produce purified plant transporters on a large-scale and rapidly discover/generate novel single-domain antibodies that can be used as valuable tools and potentially transformative and enabling resources for the greater plant science community. In addition, CROPS will determine the molecular structures of key plant membrane transporters and their natural variants, providing a new framework for understanding their functions in the context of their in planta roles, underpinning agriculturally important traits in diverse crop species.

Our understanding of plant transporters has increased dramatically over the past quarter century. This has led to a growing awareness that plant transporters play important roles in many agronomic traits, such as efficient acquisition/use of water and nutrients and crop tolerance to adverse soil environments arising from salinity, acidity, and the presence of heavy metals. Natural genetic variation is the foundation for breeding a wide variety of agriculturally relevant traits. Understanding how genetic variation translates into transporter structural and functional changes is critical to provide for a more efficient exploitation of this variation in crop improvement. CROPS will focus on key questions regarding agronomic traits that relate to food security and sustainability under marginal conditions and human health/nutrition and food safety. Specifically, through external collaborations and internal expertise, CROPS will target key transporters including those involved in micronutrient acquisition, aluminum tolerance, phosphorous acquisition efficiency, nitrogen acquisition efficiency, salt tolerance and pathogen and insect resistance. As a first step, CROPS will establish an efficient pipeline based on proven technologies for the production of plant membrane transport resources for the plant scientific community. These resources include large quantities of purified plant transporter proteins, a powerful in vitro production platform for nanobodies, novel synthetic single-domain antibodies, and state-of-the-art structural determination techniques. CROPS has a strong commitment to providing access to all data and biological resources to the broader plant membrane transport community. Access to proteins, antibody reagents, and information about protein structure will be provided through a project website/database and by dissemination through long-term repositories. Specifically, all structural x-ray and EM structural data (GC and MS) will be deposited in the Protein Data Bank (http://www.pdb.org) and the EMDataBank (http://www.emdatabank.org). All plant and synthetic nanobody DNA sequences will be annotated and deposited in GenBank and Gramene. Plasmid constructs will be deposited in Addgene (https://www.addgene.org), a not-for-profit plasmid repository. With regard to outreach, CROPS will provide mechanisms for inviting the wider community to tap into its pipeline as a basis for their own research. Researchers will be invited to submit nominations for specific plant transporters of interest. Graduate students and postdocs will receive training and access to some of the most state-of-the-art techniques in rapid antibody (nanobody) evolution, membrane protein structural/functional biology, and molecular-based trait analysis for advancing independent careers. CROPS will have a strong emphasis on providing research training and laboratory experiences to high school and undergraduate students from underrepresented groups, with a focus on development of writing and presentation skills as well as mentoring students to take personal responsibility and gain scientific educational and project management skills.

Project Summary Neuromodulators play an important role in our life by dynamically adapting the brain to an ever-changing environment. By simultaneously activating multiple receptor classes that are distributed amongst distinct components of neural circuits, neuromodulators can effectively rewire the brain in a reversible manner. However, the involvement of specific neuromodulators in distinct cognitive processes has been difficult to establish. Neuropeptides, in particular, are a unique and mysterious class of neuromodulator that are released from neurons that also release classic neurotransmitters such as glutamate or GABA. The behavioral contexts and neural activity patterns that drive neuropeptide release have been difficult to establish experimentally due to a lack of spatiotemporally precise tools for monitoring neuropeptide levels in the brain. Our current understanding is based largely on studies that rely on microdialysis for detection and pharmacology to interfere with or mimic neuropeptide signaling in behaving animals, leaving the following fundamental questions unanswered: When and where are neuropeptides released in the brain during distinct behaviors? Once released, how far do they spread from release sites, and how long do they remain to influence neural activity? To address these and related questions, we propose to engineer Nanobody-Evolved Optical Neuropeptide Sensors (NEONS) that will report the presence of neuropeptide in the extracellular space. NEONS will readily interface with contemporary imaging technology to provide highly sensitive, spatially and temporally precise monitoring of neuropeptide release in the brain. Our approach relies on the ability to evolve nanobodies in the laboratory that bind to neuropeptides with high affinity. Uniquely, our strategy is to engineer two nanobodies that form a ternary complex in the presence of neuropeptide and to translate complex formation into an optical signal using fluorescent proteins. In Specific Aim 1 we will implement a novel, powerful method for directed evolution of nanobodies developed in the Chang Laboratory called GAIN recombination. In Specific Aim 2 we will engineer these dual-binding affinity elements into optical sensors by fusing them with fluorescent proteins that function as FRET pairs and by tethering the nanobodies to circularly permutated GFP. The resulting NEONS will be optimized and characterized in brain tissue in Specific Aim 3 using fluorescence microscopy. NEONS promise to enable large-scale, potentially whole brain imaging of neuropeptidergic transmission in behaving animals. Such unbiased experiments will reveal otherwise invisible roles for neuropeptides in specific brain regions and will motivate further studies that dissect the mechanisms by neuropeptides transform circuit function to regulate behavior.

Project Summary/Abstract A major focus of the UC San Diego Superfund Research Program Center (SRC) is on the impact of toxicant exposure on the development of liver cancers and fibrosis with an emphasis on Toxicant-Associated Steatoheptatitis (TASH). As part of this project, we focus on the role of several hazardous substances on the Priority List of Hazardous Substances by the Agency for Toxic Substances and Disease Registry (ATSDR). Current analytical techniques used for the detection of such pollutants/toxicants are generally expensive to use and typically done in the lab setting. Clearly, there is a great need for cheap biosensors for specific detection of toxicants that is: (1) inexpensive to produce, (2) easy to use, (3) portable/deployable in the field, and (4) a game- changing research tool for studying environmental health sciences. We have, therefore, developed an innovative technique that will make it possible to detect and quantitate metals and other environmental toxicants by creating genetically encodable molecular sensors. Using a new and powerful molecular evolution platform, we combine non-homologous site-to-site recombination on modest sized libraries with selection strategies to discover protein biosensors that light upon addition of a specific ligand or small molecular weight toxicant. These biosensors can also be made to become molecular switches that will turn ?ON? and light/fluoresce with desired color/wavelength upon binding as well as reversibly turn ?OFF? when the small molecules diffuse away, providing a dynamic measure of its concentration. Protein biosensors and switches will allow us to track toxicants such as metals (As, Cd, Pb, Hg) and other ubiquitous environmental agents such as organochlorides, polycylics, and dioxins. We envision developing panels of inexpensive small-molecule biosensors that can be spotted on filter paper, for example, to do rapid on-site detection of toxicants and metals.

PROJECT SUMMARY Prenatal exposures to environmental chemicals have been shown to cause adverse later life health effects, often involving disorders of reproductive dysfunction. The overall goal of this research is to understand the mechanisms governing accumulation of environmental chemicals in the embryo, so that we can predict and mitigate the negative effects of these exposures. In this proposal, we address two key questions with regard to xenobiotic accumulation in the embryo, with a specific focus on the role of xenobiotic transporters during primordial germ cell (PGC) formation. First, we ask how the program of development leads to changes in xenobiotic transporter expression, and thus generates windows of susceptibility or resistance to xenobiotic accumulation. Second, we ask how real-world chemical mixtures, containing both substrates and inhibitors of transporters, impact the efficacy of this conserved, protective system. Aim 1 uses a powerful in vitro molecular evolution technology to rapidly evolve, validate, and use antibody-like binders called nanobodies to characterize xenobiotic transporter proteins in human PGC-like cells (PGLCs) and in model organism embryos (sea urchin and zebrafish). Aim 2 applies biochemical and cellular approaches to determine relevant environmental ligands of human and model system xenobiotic transporters, and takes advantage of a powerful molecular structure determination pipeline to dissect the molecular mechanisms of these interactions. Aim 3 uses models and molecular targets from Aims 1 and 2 to test the hypothesis that PGCs are vulnerable to the interfering effects of environmental chemicals on the transporter defense system, and that disruption of this system leads to decreased reproductive fitness after xenobiotic challenge. This results will provide new insights into how environmental and developmental factors act in combination to govern the susceptibility of the nascent embryonic germ line to teratogens.

PROJECT SUMMARY/ABSTRACT Down syndrome (DS) is the most common chromosomal disorder with an occurrence of 1 in 700 births. It is, by far, the leading known cause of intellectual disability. The longevity of DS people has thankfully increased and several health issues have subsequently emerged, sharing commonality with Alzheimer?s disease (AD). DS is caused by an additional chromosome to the normal 21st pair (Trisomy 21). Chromosome 21 encodes several genes contributing to AD, including proteins that phosphorylate tau, which causes tauopathy in the brain and eye. Multiple ocular anomalies, including cataracts, occur at a much higher frequency starting at younger ages for people with DS. These ocular disorders are associated with A? and also tau. To carefully understand the contribution and role of tau in the eye, we adapt a technology platform for the discovery of a panel of nanobodies (Nbs) for A? in our parent grant that can be used as specific probes to investigate the histological distribution of tau in the eye (retina and lens tissue). These Nbs would allow us to identify overlooked and potentially rare tau forms and their localization in DS eyes, perhaps yielding also some important insights for AD. For this supplement, our specific aims mirror those of our parent grant for tau: (1) Discover, produce, and validate Nbs against different conformations of tau forms and (2) Test and validate our panel of Nbs using eye tissues and lens from AD mouse models recapitulating DS.

Harmful algal blooms (HABs) threatening ecosystems, fisheries and human health worldwide, are driven by one diatom genus, Pseudo-nitzschia, along with several dinoflagellate genera and cyanobacteria. In the last decade, various species of Pseudo-nitzschia have produced devasting blooms of the neurotoxin, domoic acid (DA), in the coastal waters of Australia, Brazil, California, Chile, France, the Gulf of Maine, Indonesia, Italy and Tunisia. Currently there is great uncertainty regarding how ocean acidification, increased concentrations of atmospheric CO2, precipitation and nutrient stress will shape the productivity, and global range of Pseudo-nitzschia species' HABs. Indeed, long term trends have emerged that link toxic Pseudo-nitzschia spp. blooms to more frequent DA contamination of shellfish fisheries in the warmer ocean temperatures that prevail during warmer years. Our collective experience with diatom molecular genetics, current diatom model organisms, marine biochemistry and microscopy and our recent discovery and characterization of the hitherto unknown pathway for DA biosynthesis places us in ideal position to establish, Pseudo-nitzschia sp., as new model diatom. In so doing, molecular biological methods will be developed to enable genome-to-phenome investigations of the regulation and biosynthesis of DA. Specifically, a method to transform Pseudo-nitzschia sp driven by bacterial conjugation will be developed and disseminated to marine labs worldwide.

Our research will establish a new model diatom, Pseudo-nitzschia sp., and produce and disseminate state-of-the-art molecular biological methods to characterize the genome-phenome linkages that drive domoic acid (DA), harmful algal blooms worldwide. Taking a two-track approach, we propose to significantly upgrade the functional genomic and molecular biological tools currently used by the diatom community. Track I, We will construct a bacteria-diatom conjugation protocol and a concomitant, maintainable episome for transgenic gene delivery in Pseudo-nitzschia australis and multistriata, two globally dispersed, highly-toxigenic diatom species. Developing and disseminating new methods for diatom transformation will increase the number, and variety of species of diatoms available for functional genomics studies. Track II, With robust genetic manipulation tools for Pseudo-nitzschia spp. in place, we will develop a new diatom toolkit for molecular and biochemical applications. The first three tools to be developed are a) nanobody-fluorescent proteins (Nb-FPs) that can either repress or activate the target protein's function in vivo; b) inducible fluorescent biosensors (UnaG) that can detect DA in laboratory or seawater cultures; c) proximity-labeling enzymes: TurboID and miniTurbo, that provide insights into protein-protein interactions in vivo. As the development of the conjugative transformation protocol for Pseudo-nitzschia spp proceeds, we will express and troubleshoot each of the proteomic tools in the model organism, Phaeodactylum tricornutum.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.