Mark D. Goulian - US grants

EIA-0130797 -Harvey Rubin-University of Pennsylvania-Modeling and Analysis of Biological and Information Networks

The overall goals of our research are to: 1) create enabling technologies and experimental systems that are necessary to understand and predict the integrated functions of two bacterial sensing and regulatory networks--porim osmo-regulation in E.coli and oxygen sensing regulation of DNA synthesis in Mycobacterium tuberculosis; 2) model and abstract principles of organization, design control and coordination of biological systems. We believe that a better understanding of networked, hybrid models in biology will provide deeper insights into networked, embedded systems. No systematic approach to designing and developing such hybrid systems exists today.

Our research on the porin osmo-regulatory system in E. coli will investigate crosstalk between the porin osmo-regulatory system and other signaling systems. We suggest that the ability of the sensing element of the system, EnvZ, to act as both a kinase and phosphatase is crucial for the control of information flow and to minimize crosstalk. We will extend our models in a related series of experiments on the PhoQ/PhoP two component systems, which responds to changes in the extracellular magnesium concentration. Since the levels of the histidine kinase PhoQ and response regulator PhoP are modulated by the concentration of phosphorylated PhoP, we will be able to establish the effect of this feedback and its influence on robustness of the overall system behavior.

This proposal was received in response to the announcement NSF 01-157.

The overall objectives of this proposal are to: a) develop novel methods and tools for design and engineering of membrane proteins and protein assemblies based upon the integration of sophisticated computational chemistry techniques with in vitro directed molecular evolution; b) engineer novel membrane pores based upon the bacterial porin OmpF for controlling membrane vesicle permeability; c) engineer novel membrane fusion machines based upon influenza virus hemagglutinin for regulating bilayer fusion and membrane protein display; d) further the understanding of the physical and chemical properties underlying membrane protein structure and activity; e) train science and engineering students in these interdisciplinary nanoscience research methods.
To incorporate stable proteins that span the ~4nm thickness of the lipid bilayer and that can mediate enzymatic functions or changes in conformation requires control of protein structure at the nanometer (and sub-nanometer) scale. In their proposal, novel membrane proteins will be constructed by combining rational design, partially random design via combinatorial libraries, and directed evolution. Two different systems will be focused on as starting points: bacterial porins, which are large permeability membrane pores, and the influenza virus protein hemagglutinin, which is a pH inducible membrane fusogen. A great deal of structural and functional data has been accumulated for both of these systems, and enormous potential exists for using them to build useful membrane based devices. Channels with altered and regulated permeability could be used to selectively deliver compounds to the ambient environment or selectively internalize and process external substrates. Similarly, gated fusogens could be used to control mixing between vesicles containing two different reactants and could also be engineered to act as switches that regulate the display of protein domains. Both gated pores and fusogens could also be incorporated into synthetic lipid assemblies in order to construct new "smart" materials, whose bulk elasticity and/or permeability are modulated in response to environmental signals.
Beyond the specific utility of the proteins that they will engineer, the tools that they will
develop and the new membrane proteins that emerge will provide valuable insight into the
relatively primitive field of membrane protein design. The engineering of soluble proteins has
burgeoned into an enormous field that is moving rapidly and is far ahead of the corresponding field for membrane proteins. In particular, the powerful tool of directed evolution, which has given rise to a wide range of new soluble proteins, has not been applied to the design of membrane-active proteins.

A grant has been awarded to explore the transmission of information through the EnvZ/OmpR two-component regulatory system in Escherichia coli. The osmo-regulation of porin expression by the sensor kinase EnvZ and response regulator OmpR is one of the simplest and best characterized two-component systems and is thus ideal for studying quantitative and mechanistic aspects of intracellular signaling. The goals of this project are oriented towards addressing the following questions: 1) Can models of EnvZ/OmpR signaling describe qualitative and quantitative aspects of porin osmoregulation in vivo? 2) What is the signal that stimulates the sensor kinase EnvZ? A quantitative model of the EnvZ/OmpR circuit has been formulated which predicts that the cycle of phosphorylation and dephosphorylation renders the system insensitive (robust) with respect to variations in a number of parameters. Fluorescent reporter strains have also been developed that permit rapid and sensitive quantitative measurements of transcriptional activity. These reporters will be used to test the predictions regarding robustness, quantify the variability in signaling behavior among single cells, characterize the kinetics of EnvZ/OmpR signal transduction, and determine whether EnvZ responds to mechanical stress. Quantitative models will be further developed and analyzed in order to explore the organization and structure of the EnvZ/OmpR circuit and to explore the extent to which variants of the models can be applied to other systems.

This project combines quantitative measurements and mathematical modeling in order to understand a relatively simple example of a biological circuit. Progress in this area will provide important steps towards developing an integrative view of the complex networks of interacting biomolecules within cells. This will ultimately make it possible to reengineer many of the adaptive signaling systems in bacteria, plants and fungi and will impact a wide range of fields such as biotechnology, drug development, and cell signaling.

Two-component signaling systems are one of the major modes of signal transduction in bacteria. These regulatory circuits, which in their simplest forms are characterized by two proteins, a sensor kinase and a response regulator, play a central role in controlling basic aspects of microbial physiology and mediate responses to diverse environmental signals. Genetics and biochemical studies have made substantial progress in establishing the mechanisms that govern signal transduction in two-component systems. However, there has been relatively little work on the spatial and temporal organization of this important class of circuits within cells. This research will focus on the EnvZ/OmpR system in Escherichia coli, which is one of the simplest and best characterized examples of two-component signaling. In preliminary work, functional fusions of green fluorescent protein (GFP) to the histidine kinase EnvZ and to the response regulator OmpR were constructed. It was found that both proteins exhibit a heterogeneous distribution within cells. The present research will study the spatial localization of these regulatory proteins, using a combination of molecular biology, fluorescence microscopy, genetics, and quantitative analysis. The work will focus on the molecular determinants and dynamics of EnvZ and OmpR localization and on the correlation with signaling activity. This research will provide the first steps towards formulating a detailed picture of the spatial organization of this simple regulatory circuit and towards understanding its physiological significance. The results will also have important implications for the cell biology of other two-component systems and more generally provide new insights into the spatial organization of the bacterial cell. The broader impacts of this project will provide valuable interdisciplinary training for graduate students, undergraduates, and high-school students in aspects of molecular biology and microbiology as well as biophysics and mathematical analysis. The research will thus train scientists who are able to effectively bridge the gaps between the biological sciences and traditionally more quantitative disciplines such as mathematics, physics, and computer science.

[unreadable] DESCRIPTION (provided by applicant): Two-component systems are one of the major modes of signal transduction in bacteria. They provide an excellent class of circuits for exploring, through mathematical modeling and experiments, many of the basic design principles underlying cell signaling. The long-term objectives of the research in this proposal are to develop and test mathematical models for the network of two-component systems in E. coli. The research in this proposal will build on previous work in which we have formulated a mathematical model for some of the simplest examples of two-component signaling and have developed a number of new experimental tools for studying these systems. The research will combine mathematical modeling with experimental tests that make use of genetics and fluorescence microscopy to follow various steps in the signaling process in live cells. The specific aims of this proposal are to: 1) Analyze the dynamics of the phosphorylation cycle in two- component signaling and properties of the circuit at steady state; 2) Analyze the role of positive autoregulation in two-component signaling to determine its effects on the steady-state and dynamic properties of the circuit and test the predictions using the PhoQ/PhoP two-component system; 3) Develop and test models for response regulator binding to DNA in vivo to infer the functional relation between the fraction of bound response regulator and transcriptional activity; 4) Develop and test a model of weak crosstalk from a separate two-component system and from other phosphodonors to test the hypothesis that the phosphorylation cycle and stoichiometry of regulatory proteins are important for crosstalk suppression. Many two-component signaling systems play important roles in pathogenesis. These include circuits that regulate the expression of virulence determinants as well as more general stress-responsive or antibiotic resistance systems that are necessary for the survival of pathogens. An improved understanding of the structure of these circuits and useful models to understand the effects of circuit perturbations will likely contribute to our understanding of virulence and survival of pathogens and aid in the development of novel antimicrobials. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal requests partial support for the 2010 Microbial Stress Response Gordon Research Conference to be held July 18-23, 2010 at Mount Holyoke College, South Hadley, MA. This extraordinarily successful biannual conference attracts diverse scientists from the biological and physical sciences with broad interests in the mechanisms that bacteria employ to sense and respond to various forms of stress. The 2010 conference will have a strong representation of stress responses of pathogens as well as stress in the context of ecology and biotechnology. Stress responses associated with antimicrobials, both their mechanisms of action and resistance, will be a major theme. In addition to a session devoted to antibiotic stress and survival, numerous talks in other sessions will cover aspects of antimicrobials and related toxins. Computational and systems-level approaches to stress-responses will also be well represented and integrated throughout various sessions. In addition to the invited speakers, speakers will be selected for short talks from submitted abstracts. This will provide an opportunity for young scientists to present their work. The organizers are also committed to the strong tradition of diversity that has characterized this meeting and will work to include women and members of underrepresented groups as speakers and participants. This meeting will play a strategic role in advancing microbial research by integrating cutting edge, genetic, molecular, and computational approaches to the study of microbial systems and their stress responses. The meeting provides an unparalleled venue to bring together scientists from diverse disciplines with the aim of uncovering the principals governing microbial survival and adaptation in stressful environments. Stress responses are critical for pathogens to colonize hosts, initiate virulence programs, and resist antimicrobial treatments. Thus, the insights and new directions stimulated by this meeting will help the community to develop new methods to fight off pathogens and improve human health. A better understanding of the mechanisms that microbes use to survive and adapt to stress will provide key insights into the means by which pathogens are able to colonize and cause disease in humans, and resist antimicrobial treatments. This will aid in the design of new drugs and therapeutic strategies for combating infection and the spread of disease.

DESCRIPTION (provided by applicant): Two-component systems are one of the primary modes of signal transduction that bacteria use to sense and respond to their environment. These circuits play a key role in enabling bacteria to adapt to diverse growth conditions, control developmental programs, and initiate pathogenic lifestyles. The prototypical two-component system consists of two-proteins, an upstream histidine kinase that is usually involved in signal detection, and a downstream response regulator, which controls the circuit output. Information flow occurs by transfer of a phosphoryl group from the histidine kinase to the response regulator. Two-component systems can deviate from this simple architecture, however, and have additional protein components or more complex phosphotransfer paths. In previous work we discovered that a small 47 amino acid membrane protein, MgrB, inhibits PhoQ activity. Since MgrB expression is activated by PhoP, the protein functions as part of a negative feedback loop in the PhoQ/PhoP circuit. PhoQ is also stimulated by SafA, another small membrane protein, whose expression is controlled by the acid-responsive EvgS/EvgA two component systems. MgrB and SafA are among a group of recently discovered small hydrophobic proteins that modulate histidine kinase function. In this proposal we will combine fluorescence microscopy to follow circuit behavior in single cells, with genetic analysis and modeling to explore the role of MgrB and SafA in PhoQ/PhoP signaling. We will determine the effect of these proteins on the dynamics and input-output behavior of PhoQ/PhoP signaling for stimulation with magnesium, antimicrobial peptides and pH, compare the behavior of the low pH response among natural E. coli isolates, characterize new input signals that modulate the PhoQ/PhoP circuit through MgrB, and study the growth defect associated with dysregulation of the MgrB-PhoQ-PhoP pathway. PUBLIC HEALTH RELEVANCE: The PhoQ/PhoP regulatory circuit is a critical system for enabling bacteria such as Escherichia coli and Salmonella to colonize animal hosts and cause disease. Progress in understanding the regulation of this system by small membrane proteins, the focus of this proposal, will further our understanding of how these bacteria resist host defenses and may aid in the development of new antibiotics and compounds that specifically inhibit the virulence of these pathogens.

DESCRIPTION (provided by applicant): This application represents the first competitive renewal for our Training in Bacteriology T32 program that currently supports 3 predoctoral and 1 Ph.D. postdoctoral trainees per year. Our program, which is based at the University of Pennsylvania and Children's Hospital of Philadelphia, includes 10 laboratories directed by well established principle investigators located on a single contiguous campus. This program has allowed us to recruit some of the best and brightest trainees from among a talented pool at Penn where students and post-docs have a very large number of microbiology labs to choose among. The organized training mission of this program has helped our efforts to collaborate and interact more effectively as a group: unlike our first application, our trainers now have several joint NIH grants, a growing number of joint publications, and a network of collaborations that involve our trainees. Compared to four years ago, our program has five new trainers with more likely to be added in the near future as several new assistant professors establish their research programs. Our research in progress meetings and seminars within the microbiology program have helped spur the rapid growth of exciting, collaborative work in bacterial pathogenesis, microbial immunopathogenesis, and bacterial metagenomics, with institutional support for deep-sequencing facilities, bioinformatics, and a germ-free mouse core playing important roles in these efforts. Because of our increasing emphasis on genomic approaches and technologies, we have updated the title of our program to Training in Microbial Pathogenesis and Genomics to more accurately reflect its breadth by including microbial genomic studies. This rapidly evolving area has been the focus of the projects chosen by three of our best recent predoctoral trainees as well as one of our predoctoral graduates who is now a postdoctoral fellow with Jeff Gordon at Washington University. In light of this, we have modified our training program to accommodate trainees who are interested in microbial genomics, adding new mentors, courses, and training activities. To date, a total of 12 trainees from 7 different laboratories have been supported by this T32, including 5 Ph.D. students, 4 M.D./Ph.D. students and 3 Ph.D. postdoctoral fellows. Of our trainees, 7/12 are been women and 3/12 are under-represented minorities. The research opportunities provided by the trainers coupled with strong institutional commitment and an extensive and well-organized training program will continue to provide excellent training in microbial pathogenesis and genomics to both students and postdoctoral fellows.

The research in this proposal will explore the genetic components that enable commensal E. coli to expand their niche in the context of intestinal dysbiosis associated with inflammation. The work will take advantage of a new E. coli commensal colonization model in mice that allows the growth of competing flora, and will focus on genes that are not required for colonization of healthy mice. The first aim will test the role of the PhoQ/PhoP signal transduction system, as well as additional two-component systems, in enabling adaptation to the expanded niche that leads to blooms during inflammation. The second aim will focus on the role of genes associated with an O-polysaccharide decoration that is detrimental to colonization under standard conditions. This aim will test the hypothesis that this decoration provides protection in the environment of the inflamed intestine. An understanding of the mechanisms that enable adaptation to dysbiotic environments and the role of blooms in imposing selective pressures on the E. coli genome will lead to a better understanding of the dynamic environment of the gastrointestinal tract and to potential strategies for controlling dysbiosis. In addition, many genes required for survival in the context of inflammation may also be important for infection by pathogenic E. coli isolates, either in intestinal or extra-intestinal environments. Thus the selective pressure to bloom during chronic or intermittent inflammation may be critical for the maintenance of genes that contribute to the virulence of pathogenic E. coli. This may lead to new therapies to combat and limit the spread of pathogenic E. coli and related bacteria.

Escherichia coli and related bacteria employ TorT/TorS/TorR signal transduction system to sense and respond to the presence of the small molecule trimethylamine oxide (TMAO) by controlling expression of the proteins that convert TMAO to trimethylamine. When oxygen is absent, this respiratory process enables E. coli to extract more energy from the surroundings than would be possible by fermentation alone. In contrast with many other anaerobic respiratory systems, TMAO respiration is also active when oxygen is present. However, under aerobic growth conditions, the output of the TorT/TorS/TorR system is highly heterogeneous across the population of cells, whereas anaerobically growing cells are much more homogeneous. As part of the long-term goal of understanding the interplay between the diverse signal transduction systems that E. coli employs to adapt to diverse environmental conditions, this proposal will explore the mechanistic basis for the oxygen-dependent regulation of cell-to-cell variability in Tor signaling as well as the physiological significance of this behavior. The first aim will test the hypothesis that the variability results from stochasticity in relative levels of the TMAO-binding protein TorT and the sensor kinase TorS, the two proteins that mediate the initial steps of sensing TMAO, and will determine the mechanism of oxygen regulation. The second aim will test the hypothesis that aerobic variability enables a subpopulation to more readily adapt to a rapid decrease in oxygen levels. TMAO respiration may be important for E. coli and other pathogens to thrive in the context of intestinal inflammation, colonization of the urinary tract infections, and possibly other types of infections. Thus, a better understanding of the Tor signaling system may enable new therapies for combatting pathogens and modulating gut flora.

This application is a new submission for our Microbial Pathogenesis and Genomics training program, which was previously supported by a T32 from 2006-2016. We are requesting support for five predoctoral trainees per year. The program, which is based at the University of Pennsylvania and Children's Hospital of Philadelphia and is located on a single contiguous campus, includes twelve laboratories directed by well- established principle investigators and three junior investigators, who are newly appointed assistant professors. Based on the success of the previously funded program, this T32 will enable us to continue to recruit some of the best and brightest trainees from a talented pool at Penn and from a vibrant biomedical environment that offers a large number of outstanding research opportunities for students. The organized training mission of this T32 has helped our efforts to collaborate and interact more effectively as a group; our trainers have several joint NIH grants, numerous joint publications, and a network of robust collaborations that involve our trainees. Our research in progress meetings and seminars within the microbiology program have helped spur the rapid growth of exciting, collaborative work in bacterial pathogenesis, microbial immunopathogenesis, and bacterial metagenomics, with institutional support for deep-sequencing facilities, bioinformatics, and a germ-free mouse core playing important roles in these efforts. These resources have been expanded to the new Penn-CHOP Microbiome Program, which will be a major focal point for new research and collaborations within our T32. The retention rate of our previously funded program was perfect; no trainee left the program prematurely over the 10 years of funding. In addition, all of our former trainees have continued to work in Microbiology or closely related fields involving infectious diseases. Five of our previous trainees have obtained tenure-track faculty positions ? all at major universities. The research opportunities provided by the trainers in this program coupled with strong institutional commitment and an extensive and well-organized training program will continue to provide excellent predoctoral training in microbial pathogenesis and genomics.

Escherichia coli and related bacteria employ multi-step phosphorelays to sense intra- and extra-cellular environmental signals and to modulate diverse cellular processes. However, the significance of the multiple phosphoryl transfer steps that characterize these systems remains poorly understood. As part of our long- term goal to understand the interplay between the various signal transduction systems that bacteria employ to adapt to diverse environmental conditions, this proposal will explore several of these systems that are particularly tractable, due to well-established inputs and/or outputs, and that play critically important roles in E. coli physiology. The primary focus will be on two phosphorelays that are important for the transition to anaerobiosis, Arc and Tor, as well as a phosphohistidine phosphatase that modulates the nitrogen-related phosphotransferase system. Progress in understanding these networks will provide new insights into both the mechanisms enabling infections by pathogens and the maintenance of a healthy microbiota in host niches. The results may ultimately lead to the development of novel antibiotics or treatment regimens, as well as strategies for manipulating the microbiomes to maintain the health of the host.