Helen Elizabeth Blackwell - US grants

This research program targets the development of new chemical methodology that will be of wide utility to the synthetic and combinatorial chemistry community and the generation of chemical tools for the dissection of bacterial communication pathways. The molecular level features that are essential for quorum sensing activation or inactivation in plant-associated bacteria will be revealed by these studies, and the chemical tools that are developed will provide new insights into our evolving understanding of plant-microbe interactions. A chemistry teaching internship program will be developed to serve as a prototype for others nationwide, with the objective of developing a general strategy to engage and train future faculty in the chemical sciences. Infrastructure, support, and publicity made available through the NSF-funded Center for the Integration of Research, Teaching, and Learning (CIRTL) and the Delta Program on the UW-Madison campus will provide a strong foundation on which to build this internship program.

This CAREER award from the Organic and Macromolecular Chemistry Program supports the research of Professor Helen E. Blackwell, of the Department of Chemistry at the University of Wisconsin. The broad goal of this research plan is the design, synthesis, and evaluation of new chemical inducers that modulate communication mechanisms in plant-associated bacteria. The 'language' that bacteria use for communication is diffusible small molecules (or 'autoinducers') and this language is perceived by their cognate protein receptors. Bacteria use this chemical language to assess local population densities in a process known as 'quorum sensing'. Plant-associated bacteria use quorum sensing to regulate critical processes both harmful and beneficial to their plant host. It is now evident that certain plant hosts, in turn, 'listen' to these bacterial signals and can respond with their own chemical signals. The elucidation of this complex prokaryotic/eukaryotic communication network would represent a fundamental scientific advance. The reliance of both bacteria and plants on a language of small molecules places organic chemists in a unique position to uncover the fundamental principles underlying this communication network and design new tools to modulate it at the molecular level. Methods to control bacterial quorum sensing in plant-associated bacteria would have a major impact on agricultural science, because >50% of crop disease worldwide is caused by quorum sensing regulated behaviors in bacteria. Further, molecules that inhibit bacterial quorum sensing represent an entirely new class of anti-infectives that could have immediate impact on human health. Professor Blackwell is also pursuing a strategy to integrate training in effective teaching practices into the graduate and postgraduate experience, driven by the concept of teaching-as-research and combining substantive teaching internships with the development of new 'active learning' instructional materials for undergraduate organic chemistry.

[unreadable] DESCRIPTION (provided by applicant): The broad goal of this project is the design, synthesis, and evaluation of new chemical inducers that modulate cell-cell communication mechanisms in bacteria. The ability of bacteria to communicate with themselves and function as a group is crucial in the development of infectious disease. Gram-negative bacteria use a chemical 'language' of small molecules (or autoinducers) and their cognate protein receptors to sense their local population densities in a phenomenon known as 'quorum sensing'. At high population densities, pathogenic bacteria use this sensing mechanism to organize into structured communities called biofilms and activate virulence pathways that are the basis for myriad chronic infections. The development of methods to control bacterial quorum sensing and attenuate biofilm formation would have a major impact on human health. We hypothesize that synthetic ligands can be used to intercept bacterial autoinducer/ receptor binding and modulate quorum sensing and biofilm formation. This strategy would allow us to address fundamental questions in the field of bacterial communication. First, the ligands we uncover will reveal the molecular level features that are essential for small molecule promotion or suppression of quorum sensing. Second, synthetic ligands could be used to probe the conformational requirements for autoinducer receptor activation and inactivation. Third, tailored higher affinity ligands would enable isolation of the numerous recalcitrant autoinducer receptors. We have developed an approach to address these questions that integrates synthetic organic, combinatorial, and biophysical chemistry techniques to rapidly identify new molecules that modulate quorum sensing in bacteria. The proposed research has three Specific Aims: (1) To design and synthesize new ligands that target bacterial autoinducer receptors, (2) To test the effects of the synthetic ligands on quorum sensing in relevant pathogenic bacteria, and (3) To characterize the binding interactions of non-native ligands with autoinducer receptors using modern biophysical techniques. We have validated this approach in our preliminary studies through the synthesis and identification of a set of new small molecule antagonists of quorum sensing. Relevance: Bacteria use chemical signals to initiate the majority of human infections. The discovery of methods to block these signaling pathways would have a profound impact on public health. There is an urgent, global need for new antimicrobial therapies; the ability to interfere with bacterial virulence by intercepting bacterial communication networks represents a completely new therapeutic approach and is clinically timely. [unreadable] [unreadable]

The Analytical and Surface Chemistry Program in the Division of Chemistry, with co-funding from the Biomolecular Systems Cluster in the Division of Molecular and Cellular Biosciences, is supporting Profs. Lloyd Smith and collaborators Helen Blackwell and Michael Shortreed at the University of Wisconsin to study the nature and significance of interactions between molecules in biological systems. Specifically, they are examining the "language" that bacteria use for communication involving diffusible small molecules (or "autoinducers") perceived by cognate protein receptors. Bacteria use this chemical language to assess local population densities in a process known as "quorum sensing" which enables regulation of critical processes both harmful and beneficial to their plant host. Methods to control bacterial quorum sensing in plant-associated bacteria would have a major impact on agricultural science, because >50% of crop disease worldwide is caused by quorum sensing-regulated behaviors in bacteria. Further, molecules that inhibit bacterial quorum sensing represent an entirely new class of anti-infectives that could have immediate impact on human health. The work aims to develop new technologies for the elucidation of this complex communication network - a fundamental scientific advance. The focus is to create a more general platform for the multiplex analysis of bioaffinity interactions and to develop and apply this platform in the context of bacterial quorum sensing. The platform combines three powerful capabilities: (1) label-free measurements of bioaffinity interactions; (2) a novel substrate, which permits versatile and stable biomolecule attachment; and (3) mass spectrometric (MS) analysis capability to identify unknown ligands binding to the surface. This combination of three powerful and synergistic capabilities in a single system will provide a tool of unprecedented utility for the analysis of bioaffinity interactions.

The research groups involved are interdisciplinary, training researchers at many different stages of their education. Current collaborations involve over 20 different groups on campuses worldwide, plus two companies, and span the biological and physical sciences. In conjunction with the research, the PIs engage in interdisciplinary undergraduate research training; graduate student professional development through the Delta program (the UW implementation of the NSF Center for the Integration of Research, Teaching and Learning program); professional development for junior and senior high-school instructors through regular lectures by UW faculty; and a day-long hands-on science experience for high-school AP-chemistry students.

With this award, the NSF Chemistry of Life Processes program is supporting the research of Professor Helen E. Blackwell at the University of Wisconsin-Madison. Professor Blackwell seeks to interrogate the mechanisms by which eukaryotes intercept bacterial communication pathways at the host-bacteria interface. Bacteria use a chemical language of small molecules to assess local population densities in a process known as "quorum sensing" (QS). Plant-associated bacteria use QS to regulate critical processes both harmful and beneficial to their plant host. It is now evident that many plant hosts, in turn, "listen" to these bacterial signals and can respond with their own chemical signals that influence bacterial pathogenesis and symbiosis. The reliance of both bacteria and plants on a language of small molecules places organic chemists in a unique position to uncover the fundamental principles underlying this communication network and design new tools to modulate it at the molecular level. Blackwell and her team propose to (1) identify non-native small molecules capable of intercepting bacterial QS at the plant-bacteria interface, (2) delineate the mechanisms by which plants sense and respond to native and non-native QS signals, and (3) characterize the native small molecule signals used by plants to intercept bacterial QS.

The Broader Impacts of this research project are potentially wide-ranging. Prof. Blackwell's discoveries should be applicable to host organisms beyond plants, and will likely shed light upon the mechanisms by which QS occurs in host eukaryotes, in general. As such, the CLP program sees this project as having elements of high risk/high reward in interrogating front-line questions in the growing fields of QS and sociomicrobiology. Students involved in this project will be exposed to multidisciplinary research involving organic synthesis, bacteriology, protein biochemistry, molecular biology, plant science and bio-analytical chemistry. This is expected to provide an excellent training environment at the chemistry-biology interface.

With this award, the Chemistry of Life Processes Program in the NSF's Chemistry Division is funding Dr. Helen E. Blackwell from the University of Wisconsin-Madison to investigate the chemical signals that bacteria use to communicate with each other. This communication pathway is important, as many common bacteria use it to initiate infections in humans, animals, and plants. This project aims to study the chemical structures of the signals used by one class of bacteria and use the obtained information to design and create signals that the bacteria cannot make themselves. These non-natural signals are used to activate and inhibit bacterial communications pathways on demand. Such a chemical approach provides fundamental new insights into the biological mechanisms by which bacteria communicate and shape basic understanding of bacterial signaling. Realization of these project goals is providing research training for graduate students in modern experimental techniques, preparing them for advanced careers in science. Another major thrust of this project is, in collaboration with an established artist, creating artwork and an immersive installation piece where the potential of bacteria to act as cooperative partners is re-imagined. This medium is being used in communicating the presence of bacteria, their chemical signaling networks, and their importance to a broad non-scientist audience in the USA and beyond.

The broad goals of this research project are to characterize the chemistry and biology of the autoinducing peptides used by Gram-positive bacteria for cell-cell communication, or "quorum sensing" (QS), and to exploit these findings for the development of new chemical tools. QS in Gram-positive bacteria depends on peptide-derived signals and is best characterized in staphylococcal species. These bacteria use agr-type, two-component signaling systems for QS that are reliant on autoinducing peptide (AIP) signals and cognate AgrC receptors. Many infection-related (i.e., virulence) phenotypes are under the control of the agr QS system. Accordingly, there is significant interest in the development of chemical and biological strategies to modulate agr-type QS signaling and thereby attenuate the impact of these pathogens. Professor Blackwell's laboratory recently discovered a suite of non-native, AIP-derived peptides that are the most potent synthetic inhibitors and activators of AgrC receptors, and thereby QS. Despite their potency, AIP-derived AgrC modulators possess physical qualities that limit their utility as chemical tools. Further, there is a lack of basic mechanistic understanding of how these ligands interact with AgrC receptors and modulate their function. This project addresses these limitations with the integrated use of synthetic chemical, biochemical, microbiological and structural biological approaches. The expectation is an expansion in the current knowledge of agr-type QS.

PROJECT SUMMARY/ABSTRACT The mission of the UW?Madison CBIT program is (i) to train graduate students to recognize and address problems that transcend the traditional boundaries of chemistry and biology using innovative and rigorous approaches, (ii) to provide professional development skills and career guidance that maximize the impact of student training, and (iii) to develop an inclusive and supportive community of engaged scholars working at the chemistry?biology interface. This renewal application requests support for 10 predoctoral trainees per year, with each trainee appointed to the program for two years. Key themes are underscored below: ! Our CBIT program will help train the next generation of the biomedical research workforce. We seek to provide cross-disciplinary research training to our students, so that biologists not only appreciate, but also use, the tools and techniques developed by chemists, and vice versa. It is these fresh approaches that can provide breakthroughs in science. We will provide students with foundational course work in chemical biology and teach our students the value of looking at research problems from alternate perspectives. ! We will give our students the professional skills to maximize their cross-disciplinary training in the laboratory. These professional tools will include guidance in scientific communication to diverse audiences, the responsible conduct of research, the rigorous analysis of scientific data, and career opportunities beyond academia. We then will provide opportunities for the trainees to advance their careers through the application of these tools, through outreach, an internship, and scientific conferences. ! We will build an engaged and inclusive community of scientists working at the chemistry?biology interface. Through regular interactions with trainees and trainers, we will leverage the strong interactions between faculty/departments on campus and create new scientific collaborations. In turn, we will build strong mentor/mentee relationships between our cohort of students and faculty via formal training in mentorship. ! We understand that a diversity of experience enriches the research enterprise. Throughout the pursuit of the CBIT program?s goals, we will fully commit to the inclusion of qualified students from all backgrounds. The next award period will build substantially on the strong foundation developed over the 25-year history of the CBIT grant at UW?Madison. We will enhance existing training elements, maintaining those features that have proven successful, modernizing others and implementing new features to ensure alignment with proposed alterations to the mission of the NIGMS T32 program, and streamlining current and new features in order to empower, rather than overburden, our trainees. The outcome of the UW?Madison CBIT program will be a diverse pool of expertly trained scientists who have the broad base of technical and professional skills necessary for them to contribute substantively to the biomedical workforce. Lastly, we will evaluate our ability to achieve this outcome through the implementation of the first quantitative assessment of the CBIT program.

PROJECT SUMMARY This R21 project will use potent chemical inhibitors of bacterial quorum sensing to develop new materials and explore innovative approaches to the attenuation of bacterial infections in skin wounds. These objectives will be accomplished by the pursuit of two focused and integrated Aims: (1) development of polymer-coated wound dressings that promote the local release of synthetic quorum sensing inhibitors that target virulence factor and biofilm production in the common and notorious human pathogen Staphylococcus aureus, and (2) characterization of the ability of these dressings to attenuate bacterial load and promote the healing and repair of wounds using a mouse model of bacterial skin infection. Bacterial infections pose persistent and costly threats in myriad health settings. These problems are now urgent because the current arsenal of conventional antibiotics has been almost completely depleted by the emergence of drug-resistant bacterial strains. The importance of the growing resistance threat and its potential impacts on society are nearly impossible to overstate; new approaches to treat bacterial infections and move beyond conventional strategies are desperately needed. One promising alternative approach to prevent unwanted bacterial colonization in wounds that does not involve killing bacterial cells is to target non-essential pathways that control virulence in bacteria. Such non- bactericidal `anti-virulence' strategies can circumvent resistance and represent a potential paradigm shift for the clearance of biofilms and the treatment of infections. Bacterial cell-cell signaling (or `quorum sensing', QS) is one of the most attractive targets in the emerging anti-virulence field because it controls many of the primary mechanisms that underlie bacterial infection, including toxin production, adhesion, and biofilm formation. Our laboratories have developed several of the most potent chemical inhibitors of QS currently known. These compounds and their ability to strongly inhibit virulence in S. aureus are the focus of this proposal. The proposed work is based on two broad propositions: (i) that inhibition of QS in bacteria can prevent undesirable behaviors that lead to infection in vivo, and (ii) that inhibition of QS in surface-associated bacteria can be accomplished most effectively through the design of surfaces and interfaces containing agents that target QS pathways. This innovative and cross-disciplinary research seeks to explore these new ideas and test hypotheses that will create a foundation for the development of anti-virulence treatments in the specific and clinically relevant?and increasingly urgent?context of preventing infections in skin wounds through the design of novel wound dressings that prevent bacterial QS and virulence. Our research plan unites a team of four established and actively collaborating investigators at the UW?Madison with a unique mix of expertise in quorum sensing, chemical biology, materials engineering, microbial pathogenesis, and clinical wound care to demonstrate the feasibility of this new, materials-focused anti-virulence approach.

PROJECT SUMMARY/ABSTRACT This MIRA proposal outlines an integrated research program at the interface of chemistry and biology focused on cell-cell communication in bacteria, or ?quorum sensing? (QS). QS has a major impact on human health, with some of the most common pathogens utilizing this sensing mechanism to regulate virulence?i.e., the ability to initiate infection?once sufficient cells have amassed to overwhelm a host. Understanding the molecular mechanisms of QS, its role in mixed microbial communities, and its impact on both acute and chronic disease remain pressing and unaddressed challenges in the field. For example, our understanding of how QS signaling molecules interact with their target protein receptors to activate or inhibit QS pathways is limited to four species in Gram-negative bacteria. Further, with an increasing awareness of the importance of microbial communities (i.e., our ?microbiomes?) to human health, it is astonishing how little we know about the role of chemical signaling between these organisms in the maintenance (or disruption) of healthy microbial consortia. As bacteria use simple chemical signals to regulate QS, synthetic chemists and chemical biologists are well positioned to address these problems and other related challenges at the molecular level. With support from the NIH over the past decade, the PI has advanced the development of synthetic ligands that modulate QS signaling systems in Gram-negative bacteria and has shown that these ligands can strongly attenuate QS- controlled behaviors in many pathogens. This past work situates her ideally to lead this research project. The overall vision for this MIRA project is to build on the PI's 12-year foundation of results and leadership in this area and apply a chemical approach to expand the understanding of QS across multiple scales?from individual QS signal:receptor interactions to signaling in a single species to signaling within mixed bacterial populations to interactions of the community with a host. We will achieve this vision through the pursuit of three broad Goals: (1) the development of new small molecules capable of strongly modulating QS in Gram- negative bacteria with high potencies, stabilities, and defined modes of action; (2) the application of these molecules and new chemical strategies to delineate the biochemical mechanisms of QS; and (3) characterization of the roles of QS in mixed microbial environments relevant to human health. These three Goals will be pursued through an integration of chemical synthesis, chemical biology, bacteriology, biochemistry, structural biology, and genomics. Studies will be performed in the PI's laboratory at the UW? Madison and with a team of committed collaborators with expertise in QS and methods critical to this project. The overall outcome of this project will be a drastically increased and rigorously tested understanding of QS in bacteria and its role in biologically significant environments, and a suite of new and freely accessible research tools for the QS field. Our findings will shape the development of new methods to treat bacterial disease and will directly impact human health.

With the support of the Chemistry of Life Processes (CLP) Program in the NSF Division of Chemistry, Professor Helen E. Blackwell of the University of Wisconsin–Madison is studying the chemical signals that bacteria use to communicate with each other. This signaling pathway is extremely important, as many common bacteria use it to cause infections and disease in humans, animals, and plants. However, little is known about these signals and how they function. This NSF project aims to study the chemical signals used by one class of bacteria, to design and use chemistry to make signals that the bacteria cannot make themselves, and apply these non-natural signals to activate and inhibit bacterial communications pathways on demand. This chemical approach will allow for the signaling pathway to be explored in new ways and in important, biologically relevant environments, and will provide fundamental new insights into how it works. This project will have additional broad impacts. It will provide ample opportunities for training students in modern scientific techniques, thereby preparing them for advanced careers in science. In addition, through collaboration with an artist, the project will provide artwork (for viewing in person and online) that will communicate the presence of bacteria, their chemical signaling networks, and their global importance to the general public.

This research project is motivated by the incredible ability of bacteria to act as a group at high cell number and initiate behaviors that can have devastating effects on humanity. This process is called “quorum sensing” (QS). Gram-positive bacteria use agr-type, two-component signaling systems for QS that are reliant on autoinducing peptide (AIP) signals. The reliance of common bacteria on a chemical language of small peptides places organic chemists and chemical biologists in a unique position to uncover the fundamental principles underlying this communication network and design new tools to modulate it at the molecular level. Professor Blackwell's laboratory is making important contributions to the development of non-native peptides and small molecules capable of either blocking or activating agr-type QS in the Staphylococci. Many questions remain to be addressed about the mechanisms and potential utility of these non-native compounds. The current project seeks to leverage this strong foundation of research and significantly expand the PI’s program to address several key challenges in the QS field over the next three years. The following three integrated and cross-disciplinary aims are to be pursued here: (1) apply chemical synthesis to develop physically robust, next-generation ligands to target agr-type QS with improved activity profiles; (2) apply biochemistry to delineate the molecular mechanisms by which these ligands interact with agr QS systems; and (3) apply modern molecular biology to advance new methods to expedite the identification and production of QS ligands (both native and non-native). Together, the results of these studies have the potential to significantly refine the understanding of agr-type QS.

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.