Jeffrey J. Tabor, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Highly differentiated multicellular organisms derive from morphologically symmetrical embryos with a single, discreet gene expression profile. This remarkable process is controlled by hierarchically organized networks of genetic regulatory elements which control the expression of the genes that drive morphological changes. These genetic networks frequently produce molecules which spread by diffusion or through cell surface signaling to regulate their own expression. Such systems enable multicellular patterning, the basis for the formation of higher order structures such as tissues and organs. Theoretical models have predicted that many patterns observed in biology can be generated by a genetic system composed of a short-ranging (local) self-activator and a long-ranging inhibitor. Intriguingly, the activator-inhibitor network is predicted to drive patterns of multicellular spots, stripes, oscillations and traveling waves under only slightly different kinetic parameters. Although many candidate gene networks have been proposed to use the activator- inhibitor strategy to regulate patterning, the complexity of biology has often precluded molecular-level validation. This proposal focuses on the construction of a synthetic, well-defined activator-inhibitor gene network capable of guiding pattern formation in populations of cells. The first step in this process is the construction of an autocatalytic component capable of guiding the radial propagation of a diffusible compound across a two-dimensional population of cells. This will be based on bacterial quorum sensing (cell-cell communication) systems. The autocatalytic propagation system will then be advanced to include an inhibitor component in order to dictate spot and stripe patterning within a community of growing cells. The inhibitor component selected will be based on an orthogonal quorum sensing system and will be freely membrane diffusible, enabling long range inhibition as compared to the less diffusible activator. The 'activated' state will be indicated by a fluorescent reporter gene and pattern formation will be monitored by time-lapse fluorescence microscopy under conditions compatible with cell growth. A computational reaction- diffusion model incorporating all engineered genetic components will be used to investigate the parameters affecting pattern formation. PUBLIC HEALTH RELEVANCE: During human development, groups of cells must function in concert to form the patterns which give rise to higher-order structures such as organs, and limbs. Errors in cellular pattern formation can result in a multitude of developmental defects as well as late onset diseases in adults. We aim to investigate the genetic mechanisms underlying these highly orchestrated pattern formation processes in an attempt to improve knowledge of natural and diseased cellular states. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): The ever-increasing number of bone defects arising from clinical disorders and trauma injuries constitutes a significant healthcare concern and is a costly source of annual expenditure. The finite supply of autologous bone grafts, incompatibility of allogeneically sourced bone, and poor integration within large defects provide the impetus for developing viable bone repair alternatives. To that end, tissue engineering strategies integrating cells, biomaterials, and/or bioactive factors have demonstrated tremendous progress toward regenerating bone. While bone morphogenetic protein-2 (BMP-2)'s osteoinductivity is frequently capitalized upon, its potency often compromises the integrity of regenerated bone. Despite advances in biomaterial- and gene- based controlled release approaches, precise BMP-2 delivery over a desired duration remains a critical challenge. Translational research strategies that improve regulation of BMP-2 expression levels could therefore spur the development of safer and more efficacious treatments for bone defects unable to heal through natural processes. Herein we propose the design of an innovative red/far red (R/FR) light- controlled BMP-2 release platform to promote osteogenesis in vitro and in vivo. Optical regulation is advantageous over currently existing platforms due to its non-invasiveness, analog tunability, and robust spatiotemporal resolution. Our technology will leverage recently developed optogenetic tools merging light- responsive protein domains Phytochrome B (PhyB) and Phytochrome Interacting Factor-6 (PIF6) from Arabidopsis thaliana with engineered gene regulatory elements. When illuminated by red (660nm) light, the PhyB/PIF6 system activates a downstream promoter, which can then be instantaneously turned off by far red (740nm) light. To our knowledge, no prior studies have investigated the use of these modules for tissue engineering applications. The long-term goal of this proposal entails the development of a cell-based controlled release platform in which the concentration and timeframe of a delivered therapeutic factor can be programmed via specific wavelengths of light. The objective is to derive a pool of stable rat mesenchymal stem cells (MSCs) harboring a R/FR light gene regulatory circuit for BMP-2 release. The central hypothesis is that R/FR light ratios will permit analog tuning of BMP-2 levels, and that the resulting optical precision will improve bone formation. In Specific Aim 1, we will engineer and characterize a BMP-2 expressing R/FR light-controlled gene regulatory circuit stably integrated in rat MSCs. In Specific Aim 2, we will evaluate the osteogenic potential of these genetically modified cells in vitro and in vivo using R/FR light ratios selected from Specific Aim 1 and different biomaterial carriers. If successful, the proposed research will yield a clinically relevant cell- based therapeutic factor release platform for bone regeneration, as well as a design framework translatable to other tissue engineering applications.

? DESCRIPTION (provided by applicant): The ability to understand and manipulate the biochemical events controlling how cells make decisions is central to the development of novel treatments for microbial infections, autoimmune diseases, cancer, and developmental defects. The bacterium Bacillus subtilis differentiates into stress-resistant, metabolically inert spores upon starvation, and activates a separate gene general stress response pathway when challenged with various stresses. B. subtilis sporulation and stress response are ideal model pathways for which to develop innovative new technologies to study and control differentiation. Though the basic regulatory interactions in the core gene circuits are known, we lack a systems-level understanding of how dynamical changes in the major regulatory proteins result in cellular information processing and the ultimate guide these large-scale cellular decisions. Our central hypothesis is that the ability to dynamically perturb and observe protein activities in gene circuis in real time will yield crucial insights about cellular decision-making and differentiation. To thi end, we propose to develop a technology for interrogating the signaling properties of the B. subtilis sporulation and stress response gene circuits that uses time-varying light signals to program exceptionally well-defined gene expression dynamics in live cells. As a first aim, we will re-engineer a green/red light-switchable two-component system we previously built in E. coli to control transcription and generate dynamical gene expression functions in B. subtilis. By moving this system from E. coli to the evolutionary distant B. subtilis, we will also gain insights on the considerations needed to move these powerful optogenetic tools to other model organisms and clinically important species. As a second aim, we will use our optical method to analyze how different rates of activation of the major sporulation regulators impact the circuit functions and resulting phenotype. We expect to unequivocally demonstrate that i) successful execution of the sporulation program requires not only proper steady-state levels, but also proper dynamics of key regulators and that ii) recently observed pulsing in the major stress response regulator are used to elicit proportional activation levels of many target genes. In the third aim, we will use or optical method to investigate the biological significance of the recently described pulsatile dynamics of the master sporulation regulator at the population and single cell levels. In particular, we will evaluate our recent hypothesis that the sporulation pulses must occur after DNA replication to ensure that spores inherit chromosomes and a prevailing hypothesis that a supra-threshold concentration of the master sporulation regulator must be reached for cells to commit to sporulation. Since the B. subtilis sporulation and stress response circuits are widely conserved among medically important spore-forming bacteria including B. cereus, B. anthracis, and C. difficile, our result will not only enable breakthroughs in the understanding of cellular decision-making, but also provide a basis for design of new antibacterial agents.

Project Summary/Abstract Aggregation of amyloid-? (A?) into plaques is a hallmark of AD and considered a primary event in AD pathologies. Transgenic variants of the rapidly reproducing nematode C. elegans expressing human A? accumulate aggregates with age and exhibit early lethality. C. elegans feed on E. coli bacteria, 10% of which escape digestion and constitute the gut microbiota. Due to the high genetic tractability of both host and microbe, C. elegans and E. coli provide a powerful model for studying the molecular mechanisms of gut microbiota-host interactions. In exciting preliminary work, co-investigator Wang has identified 14 E. coli genes that protect against A? induced lethality in transgenic C. elegans. We have determined that four are linked to production of the extracellular polysaccharide Colanic Acid (CA). Furthermore, we have shown that pure CA protects against A? toxicity when delivered with live bacteria. The next step is to identify the mechanism by which CA and the remaining 10 genes protect against A? toxicity. Such results would inform future studies in mammals and the engineering of therapeutic bacteria that prevent or treat AD. The major current limitation in studying the mechanisms of gut microbiota-host interactions is the lack of technologies for externally manipulating bacterial gene expression in vivo. Traditional chemical effectors of bacterial gene expression are insufficient due to complications arising from delivery, transport, and degradation. Optogenetics is a rapidly advancing technology combining light and genetically-encoded photoreceptors to control molecular biological processes in live organisms. Light can be controlled with exquisite precision in the wavelength, intensity, spatial, and temporal dimensions, affording unmatched levels of control. Previously, P.I. Tabor has transported light sensing two-component histidine kinase signal transduction systems from cyanobacteria into E. coli, and used them for unprecedented quantitative, spatial and temporal control of gene expression in vitro. The goal of this proposal is to combine P.I. Tabor's and co-I Wang's methodologies to characterize how gut bacterial gene expression affects A? toxicity in the C. elegans model. We will achieve this goal through two Specific Aims: Demonstrate precise optogenetic control of the expression of E. coli genes that protect against A? toxicity in the gut of live C. elegans (Aim 1), and characterize the relationships between the quantitative, spatial and temporal pattern of the expression of E. coli genes in the gut and the amelioration of A? toxicity (Aim 2). By achieving these aims, we will demonstrate the first optogenetic control of microbiota function in a live animal. This proposed research will directly improve scientific knowledge in gut microbiota-host interactions, and molecular mechanisms of AD pathologies. Optogenetics has revolutionized neuroscience, and is now enabling major breakthroughs in cell biology and systems and synthetic biology. The research proposed here will bring optogenetics to the fundamental and timely problem of the microbiome.

Project Summary/Abstract The human immune system produces at least 140 different antimicrobial peptides (AMPs) to kill invading bacteria. However, pathogenic bacteria use specialized pathways called two-component systems (TCSs) to detect these AMPs and activate the expression of AMP-resistance and virulence genes. This response enables pathogens to survive immune attacks and mount deadly infections. Therefore, elucidating the mechanisms by which peptides interact with TCSs is critical to understanding how infections progress. This knowledge could also lead to the design of new antimicrobial drugs that interfere with TCS-mediated AMP sensing. Gram-negative Enterobacteriaceae, such as the common pathogen Salmonella Typhimurium, cause 200,000 infections and 10,000 deaths in the United States each year. The most important AMP-sensing TCS in Gram-negative Enterobacteriaceae is named PhoPQ. Here, the membrane bound histidine kinase PhoQ senses AMPs and responds by phosphorylating the cytoplasmic response regulator PhoP, which activates a gene expression response. Though its interactions with a small number of model AMPs have been characterized, little is known about the broader peptide binding and sensing capabilities of PhoQ. The major limitations have been the cost and time required to chemically synthesize peptides and characterize their effects on TCSs using traditional microbiological or biochemical methods. In preliminary work, we have developed a new technology named SLAY-TCS that combines bacterial peptide display, fluorescence-activated cell sorting, and next-generation DNA sequencing to measure how S. Typhimurium PhoQ responds to millions of peptides in a single experiment. Using SLAY-TCS, we have already revealed that PhoQ senses a far wider range of peptides than previously known. Here, we propose to use SLAY-TCS to characterize how S. Typhimurium PhoQ responds to nearly every AMP produced by the human immune system, and thousands of mutants thereof, in order to reveal the identities, sequence motifs, and biophysical properties of PhoQ-activating peptides (Aim 1). We will also combine this approach with PhoQ mutational analyses to reveal how PhoQ sensing specificity has evolved across diverse pathogens, which may have enabled them to adapt to different biogeographical locations in vivo (Aim 2). Finally, we will use SLAY-TCS to perform the first large-scale characterization of peptide inhibitors of PhoQ, and explore the efficacy of the strongest inhibitors we identify in preventing S. Typhimurium virulence in primary mouse macrophages (Aim 3). The work in Aim 3 will reveal mechanisms by which exogenously-delivered peptides can inhibit PhoQ, and could lead to the design of novel antimicrobial therapeutics based on modified peptides in the future. Taken together, this proposal will substantially enhance our understanding of how a dangerous family of bacteria causes infections in humans and accelerate the design of sorely-needed antimicrobial therapeutics. Finally, our approach could be extended to other peptide-sensing TCSs beyond PhoPQ in future studies. 1