Mark Gomelsky - US grants

The numerous conserved protein domains of unknown function have been revealed through microbial genomics, which suggests that significant areas of microbial physiology, metabolism and behavior remain unexplored. This research is aimed at investigating functions of proteins containing GGDEF and EAL domains that are broadly distributed in diverse bacteria. The GGDEF and EAL domains are involved in synthesis and hydrolysis of the unusual nucleotide, cyclic diguanylate, c-di-GMP. The main hypothesis of this project is that c-di-GMP is an important underappreciated second messenger in bacteria. The long-term goals of the project are to elucidate the role of c-di-GMP and to reveal the mechanisms by which it operates using the metabolically versatile proteobacterium, Rhodobacter sphaeroides, as a model. Preliminary data indicate that R. sphaeroides contains enzymes involved in both synthesis (diguanylate cyclases) and hydrolysis (phosphodiesterases) of c-di-GMP. Initial emphasis of this project will be placed on two R. sphaeroides c-di-GMP-specific phosphodiesterases, designated BphG and BphH, which contain phytochrome-type domains and are likely to represent photoreceptors of unique structure. The depletion of the intracellular pool of c-di-GMP in R. sphaeroides results in specific changes in the R. sphaeroides transcriptome. Uncovering mechanisms by which c-di-GMP affects gene expression is another aim of this project. A combination of molecular genetic, transcriptomics and biochemical approaches will be employed to address these questions.

This research will fill an apparent void in our understanding of the bacterial cell resulting from an oversight of a potentially important second messenger, c-di-GMP. Elucidation of the role of c-di-GMP is anticipated to have a significant effect on the field of signal transduction in various bacteria, including agriculturally, environmentally and medically important species. This project will provide ample opportunities for interdisciplinary training of high school, undergraduate, graduate and postdoctoral students.

Cyclic dimeric guanosine monophosphate, c-di-GMP, is a key molecule involved in the lifestyle switch in bacteria, from single motile cells to surface-attached multicellular communities (biofilms). c-di-GMP controls this lifestyle switch by affecting bacterial motility, adhesion to surfaces, synthesis of extracellular matrix, stress resistance, and virulence. Gaining mechanistic insights into how c-di-GMP functions is of fundamental importance in bacteriology. Currently, a glaring gap exists between the large numbers of enzymes involved in c-di-GMP synthesis and hydrolysis (several dozen in most proteobacteria) and the small numbers (low single digits) of known c-di-GMP receptors, or effector proteins. This gap indicates that many c-di-GMP receptors have escaped identification. The long-term objective of this project is to identify new c-di-GMP receptors and uncover molecular mechanisms through which they operate in a model bacterium, Escherichia coli. In this project, new c-di-GMP receptors will be identified by genetic means as well as by the affinity-based capture. Because c-di-GMP, like other second messengers, works though conserved molecular mechanisms, it is expected that the insights derived from E. coli will inform strategies on controlling behavior of various bacterial species of medical, agricultural, environmental and biotechnological significance.

The broader merit of this proposal lies in understanding the mechanisms that control bacterial behavior. This understanding is critical for designing pharmacological interventions that would allow us to manipulate bacteria in desirable ways. This project will offer interdisciplinary training opportunities in bioinformatics, bacterial genetics, protein-ligand biochemistry, and physiology to graduate and undergraduate students who will comprise the core research personnel. It will also provide research experiences to minority high school students enrolled in the Summer Research Apprenticeship Program. The project results will be presented at scientific meetings and published in research papers as well as in the articles for broader audiences.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Engineered photoregulated proteins have the potential to revolutionize biomedical research. In a photoregulated protein, a photon absorbed by a chromophore bound to a photoreceptor protein domain affects activity of an output domain. Infrared light is harmless to mammalian cells, therefore, it can work as a highly specific, and cheap way to regulate protein activities. The spatiotemporal resolution that can be achieved by using photoregulated proteins is unprecedented as a laser beam can be focused not only on an individual cell but on a particular region of the cell. Engineered photoregulated proteins can be broadly used for activation (or inactivation) of proteins of interest in cell cultures, tissues and animal models. Thus far only blue-light photoreceptors have been used for protein engineering. Bacteriophytochromes absorb red/near infrared light, which has much higher tissue penetration capacity than blue light and is currently used in deep-tissue phototherapies. The objective of this application is to provide the proof of principle that a chromophore-binding module of bacteriophytochromes can be used for engineering of red/near infrared light regulated proteins. The goal of this pilot project is to engineer a near infrared light activated adenylate cyclase (cAMP synthase). The critical role of cAMP in controlling glucose and lipid metabolism as well as neuronal activity makes adenylate cyclase a highly desired target. The design of photoregulated enzymes relies heavily on computationally-intensive bioinformatics approaches that involve analysis and modeling of protein structures and dynamics.

DESCRIPTION (provided by applicant): The following contains proprietary/privileged information that M. Gomelsky requests not to be released to persons outside the Government, except for purposes of review and evaluation. PROJECT SUMMARY: Small molecule activators and inhibitors of signal transduction pathways are biologically useful, but are limited by their target specificities and spatiotemporal resolutio in vivo. A recently emerged optogenetic strategy can supplement chemical/pharmacological approaches. Ontogenetic involves introduction into cells and animals of genes encoding proteins whose activities can be photoactivated. Light is a unique stimulus in that it can control protein activities in vivo in a reversible manner and with spatiotemporal precision unattainable by chemicals. Photoreceptors of the bacteriophytochrome type absorb near-infrared light, which has superior tissue penetration properties, thus allowing protein photoactivation from unobtrusive external light sources. This is particularly important for studies on whole animals, such as mice. Recently, progress has been made in transplanting natural photoreceptor modules to control heterologous protein activities. Our long-term objective is to elucidate principles of engineering near-infrared light activated proteins using photosensory modules of bacteriophytochromes. This exploratory proposal will test the hypothesis that bacteriophytochrome photoreceptor domains can activate diverse homodimeric output activities. We will exploit our earlier studies of the BphG protein, a unique, near-infrared light activated diguanylyl cyclase. The nucleotidyl cyclases will be used as engineering targets. And an executioner (effectors) caspase rationally design bacteriophytochrome-based proteins, we will employ a multiprong approach involving circumventing our present inability to computational and structural analyses of proteins with genetic screening in E. coli. The proposed concept of engineering near-infrared light activated homodimeric proteins and the multidisciplinary approach to bacteriophytochrome engineering are innovative and feasible, as we already have constructed the first photoactivated homodimeric enzyme with a heterologous activity . Upon completion of this project, we anticipate to advance our understanding of the mechanism of light-induced signal propagation in bacteriophytochromes, and to uncover engineering principles for constructing homodimeric near-infrared light activated proteins. Because a large number of signaling proteins function as homodimers, light-induced protein homodimerization can be used to control a variety of cellular functions including apoptosis, differentiation, proliferation, transformation and adhesion. This research is significant because cAMP and cGMP control many cellular processes including growth, blood glucose levels, cardiac contractile function, and learning, memory and cancer cell survival. Photoactivated executioner caspase generated here will allow researchers to conduct targeted cell/tissue killing in whole animals using a mild and noninvasive procedure. These tools will likely find applications in cell biology, immunology and developmental biology, and potentially in cancer gene therapy.

DESCRIPTION (provided by applicant): Once genetically engineered cells, such as stem cells designed to repair damaged tissues, immune cells programmed to recognize and destroy tumors, or hormone-producing cells for endocrine disorders, are delivered into a mammalian host, they become poorly controllable. This situation poses unprecedented risks associated with the predisposition of the engineered cells to transformation and/or malfunction. Drugs cannot distinguish between properly functioning and malfunctioning cells inside the body, while genetically build-in safety mechanisms may prove insufficient. Optogenetic approaches to control biological processes offer spatiotemporal resolution unmatched by chemicals, yet UV-visible light cannot reach deep mammalian tissues. In contrast, light in the near-infrared window is known to be safe and penetrate mammalian tissues to the depth of several centimeters, at least several-fold deeper than UV-visible light. Therefore, light from externally placed lasers or light guides inserted into body cavities can reach internal organs and control biological activitie. Bacteriophytochromes are the only class of photoreceptor proteins that sense near-infrared light. Bacteriophytochromes autocatalytically bind their chromophore (biliverdin) that is naturally made in mammalian cells. This fortunate circumstance obviates the need for exogenous chromophore supply. In this project we intend to engineer bacteriophytochrome-based genetic modules for orthogonal gene regulation in mammals, including humans. We plan to optimize these light-activated modules for several mouse tissues. The optogenetic tools developed here will allow researchers to execute conditional and reversible gene knockouts (or gene activation) in specific tissues of live animals, which will deepened our understanding of progression of various diseases, improve our knowledge of mammalian development, and offer real-time insights into host- pathogen interactions. These tools will also make gene and engineered cell therapies safer and smarter.

Gomelsky, M, PI SUMMARY Listeria monocytogenes is one of the most problematic causative agents of foodborne illness. Despite a relatively low number of cases, listeriosis is ranked as the deadliest and the fourth costliest foodborne disease in the USA among all diseases caused by bacterial, parasitic and viral foodborne pathogens. We have characterized a major regulatory system involving the second messenger, c-di-GMP, in L. monocytogenes and found that it plays unexpectedly important roles at the interface between the saprophitic and the pathogenic lifestyles of this bacterium. One of the consequences of elevated intracellular c-di-GMP levels is severe inhibition of mammalian cell invasion. This project is aimed at (i) deciphering the mechanism of c-di-GMP- dependent inhibition of cell invasion, which appears to involve key regulators of biosynthesis and virulence, and (ii) identifying natural compounds that strongly affect intracellular c-di-GMP levels in L. monocytogenes. This project is expected to shed light onto the interplay between c-di-GMP signaling, metabolism and virulence in L. monocytogenes that is emerging as a novel regulatory paradigm. Results of this project may also lead to identifying natural compounds that inhibit listerial invasiveness and therefore may be used to reduce frequency of listerial infections.

This award will support early career researcher attendance at the Optogenetic Technologies and Applications Conference, which will be held in Boston, MA on December 8-10 of 2019. The conference aims to promote collaborative, interdisciplinary research by bringing engineers and life science researchers together at the same venue. The conference is designed to be highly interactive, with talks, poster sessions, panel discussions and informal meetings. The conference is organized into sessions on optogenetic technologies, optogenetics in biomedicine and optogenetics in bioengineering. There will be ample opportunity for sharing the latest research results and for networking.

Optogenetics is an emerging technique that allows researchers to use light in order to control living cells that have been modified to be light sensitive. Optogenetics has tremendous potential for use in research, medicine and industry. The Optogenetic Technologies and Applications Conference will be used to showcase the novel and cutting-edge research conducted in this field. The conference aims to provide a venue for the following: (1) scientific presentations from a diverse group of researchers, including biologists, chemists, and engineers; (2) bringing together members of industry and academia to showcase different perspectives and approaches, as well as to encourage potential collaborations; (3) encouraging active participation of graduate and post-doctoral researchers who represent the future of the field; (4) bringing scientists and engineers together to discuss their disciplines and approaches, and to motivate new research into barriers limiting the development of optogenetics in a variety of fields; (5) scientists who are from underrepresented groups to present their research and to network.

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.

Diabetes is a chronic disease afflicting over 30 million people in the United States. Both type 1 (childhood onset) and type 2 (adult onset) diabetes are associated with the deficiency or impairment in beta-cells in the pancreas. These are the only cells in the body that secrete insulin, an essential hormone for blood glucose regulation. Engineered insulin-producing cells can reconstitute the impaired pancreatic beta-cell function, however, the large number of required cells poses challenges on the implantation device size, and supply of nutrients and oxygen, which results in rapid deterioration of implanted cells. This project aims to enhance insulin production in engineered beta-cells via optogenetic means, i.e., using a combination of genetics and optics. Human beta cells will be genetically engineered to produce more insulin when activated by near-infrared window (NIRW) light. NIRW light penetrates deep through tissues and can reach transplanted beta-cells better than light of other wavelengths. The enhanced function of beta-cells means that fewer cells need to be transplanted to correct high blood glucose. Once developed, the technique will be tested in a mouse model of diabetes. The knowledge generated will inform the development of the next generation of technologies for efficient diabetes treatments. Educational activities, which are intertwined with the proposed research, will provide ample training opportunities for a new generation of high school, undergraduate and graduate students in STEM fields. The knowledge and associated technologies generated through this work will be disseminated to the scientific community and the general public through online media, presentations, and publications.

The goal of this project is to engineer human beta-cells with enhanced glucose-stimulated insulin secretion (GSIS) in response to near-infrared window (NIRW) light. The project capitalizes on two recent advances from the collaborating laboratories. The first advance is demonstrating that rodent beta-cells expressing a blue light-activated adenylate cyclase (bPAC) for modulation of cAMP levels significantly (~3-fold) enhanced GSIS with no increase in oxygen consumption rate. After bPAC--cell transplantation, diabetic mice subjected to blue light display improved glucose tolerance, lower hyperglycemia and higher plasma insulin. The key deficiencies of the bPAC-prototype were limited activation due to poorly penetrating blue light, nonspecific cAMP effects due to intracellular mislocalization of bPAC, and the lack of human Beta-cells. The second advance is a recently engineered adenylate cyclase activated by NIRW light (NIRW-AC) that will help overcome these deficiencies as it penetrates more deeply through mammalian tissue than blue light. The Research Plan is organized under three objectives. OBJECTIVE 1 is to design a NIRW-AC with low dark activity, improved photoactivation range and optimized expression in human beta-cells. OBJECTIVE 2 is to engineer a light inducible system for localized, target-specific photoactivation of cAMP levels. The localization of the NIRW-AC will be tuned to mirror the localization of native ACs in beta-cells thereby reducing the nonspecific effects of cAMP and improving the long-term optogenetic performance of beta-cells. OBJECTIVE 3 is to generate human beta-cells with NIRW light controlled enhancement of their GSIS. This will encompass the biochemical and functional characterization of the engineered human beta-cells expressing the optimized NIRW-AC. OBJECTIVE 4 is to test the performance of the NIRW-AC-expressing cells in a murine model of diabetes. The project?s deliverable will be a NIRW-AC with superior photoactivation, optimized spatial positioning and expression in human beta-cells for optogenetically enhanced GSIS.

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.

ABSTRACT During sleep, the thalamus generates a characteristic brief pattern of 8-15 Hz electroencephalographic (EEG) waves that predominantly occur during light stages of non-rapid eye-movement sleep (NREMS). Reduced spindle may cause impaired learning and Mild Cognitive Impairment (MCI) in AD and is a biomarker for early AD-related changes in brain dynamics. Conversely, promoting sleep oscillations by transcranial stimulation enhances memory consolidation in MCI. By developing a set of novel, noninvasive, bacteriophytochrome-based optogenetic tools to control cAMP synthesis (adenylate cyclase, AC) and breakdown (phosphodiesterase, PDE), we will make spindles accessible for noninvasive manipulations that spare other sleep rhythms. These enzymes are activated by light in the so-called near-infrared optical window (NIRW). The NIRW light-activated modules are suitable for the rapid yet long-lasting and noninvasive manipulation of cAMP in thalamic neurons in intact animals, because NIRW light penetrates through mammalian skulls and brain tissues better than the light of any other spectral region. We will examine a provocative novel hypothesis that cellular pathology and cognitive decline caused by Alzheimer?s disease (AD) related mutations can be restored via enhancing thalamocortical spindles waves during sleep in vivo. We will first develop novel noninvasive optogenetic tools to manipulate AC and spindle oscillations (Aim 1). Then, we will examine whether NIRW-AC and NIRW-PDE bi-directionally modulate the progression of AD?related neuropathology and cognitive decline via their actions re: spindle wave regulations (Aim 2). Upon completion of this project, we will have developed genetically encoded NIRW-light activated tools, allowing noninvasive manipulation in deep brain regions of live animals. Results are expected to provide a sound basis for investigation in disease models that involve spindle wave and cAMP aberrations, such as AD, and suggest novel non- invasive intervention strategies to counteract brain dementias caused by AD.

PROJECT SUMMARY Attenuated and avirulent strains of Listeria monocytogenes (Lm), that are delivered via intravenous injections, accumulate and propagate in primary tumors and metastases while being quickly cleared from healthy tissues. We intend to use these strains as remotely controlled, tumor-specific anticancer payload delivery vehicles, bactodrones. In this project we will engineer Lm to synthesize and secrete cyclic dinucleotides (c-di-NMPs) as potent innate immune system stimulators inside tumor microenvironments. On-site accumulation of c-di-NMPs will induce production of type I interferon via the STING innate immunity pathway. This will improve the capacity of Lm to induce immunogenic tumor cell death and lead to the release of tumor-associated antigens, which will facilitate recruitment of tumor-specific CD8 T cells. The sustained tumor-localized c-di-NMP production will keep T cells and other anticancer immune cells activated. To assess feasibility and efficacy of delivering intratumoral c-di-NMP via genetically engineered Lm, we will pursue two aims. In aim 1, we will engineer Lm to secrete enzymes for c-di-NMP synthesis in immune and tumor cells. In aim 2, the engineered Lm will deliver plasmids encoding a c-di-NMP synthases, via a process known as bactofection. Both approaches are expected to turn infected cells in the tumor microenvironment into c-di-NMP producing factories and ensure durable STING activation. Importantly, Lm-mediated c-di-NMP delivery systems will be made inducible with a benign chemical inducer, which will enable temporal control of STING activation and limit toxicity associated with systemic c-di-NMP exposure. Following optimization of the Lm bactodrones in vitro, and in breast cancer cell line, we will test efficacy of periodic bactodrone injections in a mouse metastatic breast cancer model. We anticipate that Lm bactodrones will become efficient vehicles for tumor-localized, temporally controlled and inexpensive delivery of genetic payloads for various antitumor activities.