Kim Lewis - US grants

The energy level of a cell is determined by the state of three interconvertible components: ATP, the proton motive force, and the redox level of the electron transport chain. Oxygen taxis, oxidizable substrate taxis and phototaxis are all behaviors governed by sensing of the energy level. This research will identify the energy-level sensor of E. coli and establish the principal mechanism of sensing. An arcB mutant fails to show O2- taxis and oxidizable substrate (proline) taxis. A cloned arcB locus will be used to identify the sensor by expressing and localizing the arcB product. By comparing cell responses to CN and uncoupler that similarly decrease the pmf, but have different effects on the redox state, it will be found whether a redox chain component or pmf per se are immediately responsible for energy level sensing. Understanding of information transduction will be achieved by isolating pseudorevertants to arcB that will map in a che gene(s). This study bridges two fields of knowledge: metabolic regulation and behavior, since arcB controls both taxis and O2-dependent transcription. It is highly possible that arcB (or a homologous compound) is additionally responsible for other instances of so-called "pmf-dependent" regulation in various bacteria, as well as in mitochondria and chloroplasts. It is of fundamental significance to learn how the bacterial cell senses its own energy level and uses this information to control behavior. A new type of sensor which is substantially different from known sensors will be explored in this study. The principal mechanism of energy level sensing will be elucidated. Since it is possible that mitochondria and chloroplasts "inherited" the energy level sensor from bacteria, this research may provide a model system for studying behavior of higher organisms.

Lewis 9317013 Addition of uncouplers to E. coli causes a low energy shock response that is accompanied by induction of multidrug resistance pumps. A Summary of the objectives aimed at identifying the components of the response and understanding their interactions is given below. 1. Expression of a target gene. Regulatory mutations in emrD, a multidrug resistance gene, that lead to constitutive expression will be used to determine the upstream site controlling uncoupler- dependent expression. 2. The signal. Expression of emrD in an ATPase mutant in response to an uncoupler will be measured to determine whether the signal originates prior to, or after ATP synthesis. Changes in pmf will be measured in parallel to determine the quantitative requirements for the inducing signal. 3. The sensor. Regulatory mutations leading to constitutive expression of emrD will be used to clone the regulator genes. It is expected that one of the genes will code for the receptor that senses uncoupling. 4. Are other MDR's target genes of low energy shock? Five more putative MDR's have been found in E. coli. the possibility that they are also induced by uncoupling will be studied using Northern blotting. Significance: it is of general importance in understanding cell metabolism to learn how the cell senses its energy status and activates a response mechanism. %%% Cells obtain energy through respiration. This is a process of oxidation of various nutrients such as sugars by oxygen. Respiration takes place in the cell membrane. The energy released in the course of respiration is stored in the form of a "high energy" molecule, ATP. A large number of toxic substances affects the membrane and leads to an "Energy Shock", or loss of energy- producing capability. We found that the cell responds to such toxins by inducing the synthesis of a special class of proteins, multiple drug resistance pumps, that extrude the toxins from the cell. How does a cell sense the presence of t oxins? What is the mechanism for the induction of the multidrug resistance pumps? These are the questions that will be studied in this project. ***

Description (Adapted from the applicant's abstract): The PI's long-term objectives are to understand the mechanism of microbial biofilm resistance to antibiotics, and to develop effective anti-biofilm therapies. This is a collaborative project that brings together the expertise of the PI in microbial multidrug resistance and the expertise and advanced genomics tools to study P. aeruginosa that have been developed by the co-Investigator and colleagues at PathoGenesis. The mechanism of extremely high resistance of microbial biofilms to antibiotics is poorly understood. Data from the literature and the PI's own findings indicate that biofilm cells might be expressing antibiotic resistance mechanisms. Finding genes responsible for biofilm resistance to antibiotics is the main goal of this application. There are two specific aims. In Specific aim 1, the PI will identify genes specifically expressed in biofilms. Two groups of experiments are proposed. (a) In vitro studies. Biofilm RNA will be isolated, labeled and used to probe a P. aeruginosa DNA array. A detailed analysis of factors likely to affect expression of biofilm genes will be performed. These factors will include the age of the biofilm, various growth conditions, including those emulating growth in vivo, and growth in the presence of antibiotics which might induce resistance. (b) In vivo studies. Sputum samples that have been collected from patients with cystic fibrosis harboring P. aeruginosa biofilm infections will be used to isolate RNA and obtain gene expression profiles. This information will allow the PI to identify biofilm-specific genes that are expressed in vivo. In Specific aim 2, the PI proposes to validate candidate genes expressed in the biofilm. Three types of experiments are proposed. (a) Mutants from an ordered Tn insertion library will be used to test involvement in antibiotic resistance of genes whose expression is changed in biofilms. Each candidate mutant will be tested in detail with a representative panel of antibiotics. This experiment will show which specific biofilm genes are responsible for increased resistance. In a complementary approach, a complete Tn insertion library will be screened for mutants with increased antibiotic susceptibility. Candidate mutants from this screen will then be tested in detail with a larger panel of antibiotics at a broad range of concentrations. (b) Possible "Universal" resistance genes will be identified by comparison with genes expressed in E. coli biofilms and probed with an E. coli DNA array. (c) P. aeruginosa mutants in genes participating in antibiotic resistance will be used to obtain expression profiles. This information will likely contribute to the understanding of the mechanism of resistance. Resistance genes identified in this project will serve as targets for drug discovery and development of effective anti-biofilm therapies.

When a bacterial cell dies, the cause of death is often autolysis, rather than direct damage produced by a harmful factor. Hydrolysis of peptidoglycan is a necessary stage in cell wall synthesis, and autolysis has been viewed as a result of "disbalance between peptidoglycan synthesis and hydrolysis", essentially a maladaptive mistake. This project explores the hypothesis that autolysis is not a mistake, but adaptive programmed death. Autolysis is part of the developmental process of fruiting body formation and sporulation in Myxococcus. Autolysis which is required for natural transformation in S. pneumoniae is another example of specialized adaptive programmed death. Bacteria show many features of complex organization, similar to multicellular organisms. Like in multicellular species, a bacterial population would benefit from eliminating defective cells. The aim of this project is to test the feasibility of the idea that autolysis is the mechanism of adaptive apoptosis in bacteria. Specifically, a search for regulatory components of a possible apoptotic pathway linking particular types of cell damage to activation of autolysins is being undertaken. A genetic approach is being used to identify putative apoptotic genes. E. coli will be mutagenized with mini-Tn10 transposon and selected for survival to lethal levels of a mutagen; and high temperature. Mutants that show resistance to killing (but not resistance to growth in the presence of) both factors will become candidates for being affected in genes coding for apoptotic compounds. "Random" PCR will be used to identify the flanking DNA regions, amplified DNA will be sequenced by single passage, and the genes carrying the Tn insertions will be identified using the database of the E. coli genome. The mutants will also be characterized phenotypically using a panel of different lethal factors. Strains with known mutations, such as those lacking autolysins, will also be tested for possible survival with the panel of lethal factors. This exploratory project will show whether E. coli has regulatory genes affecting autolysis and will lay the basis for future detailed studies of apoptosis in bacteria.

Our long-term objective is to understand the function and mechanism of microbial multidrug resistance pumps (MDRs). The main goal of this project is to identify natural substrates and inhibitors of microbial MDRs. According to our findings, berberine alkaloids are natural MDR substrates. Plants making berberine alkaloids also produce at least two MDR inhibitors (5'-methoxyhydnocarpin and pheophorbide) that act in synergy with berberine. We will use this model for an expanded search of new MDR substrates among plants producing antimicrobials, and for novel MDR inhibitors that are likely to accompany them. Our main focus will be on plants that make non-cationic antimicrobials that can be extruded by broad-specificity MDRs of Gram negative bacteria and yeast. This is a collaborative project that brings together the expertise of the PI in microbial multidrug resistance and the expertise of the co-PI in natural products chemistry. The overall project will flow from identifying antimicrobials that are MDR substrates, to finding MDR inhibitors in plants that produce these antimicrobials, to characterizing the interaction of these substances with microbial MDRs. The Specific Aims of this proposal are: 1. Identifying MDR substrates among plant antimicrobials. Plant antimicrobials will be screened with a panel of Gram positive bacteria, Gram negative bacteria and yeast in search of MDR substrates. We will screen a large collection of substances, representing the main types of antimicrobials (alkaloids, flavones, quinones, phenols) coming from a variety of unrelated plants. Antimicrobials that are more active against strains lacking MDRs are likely MDR substrates. 2. Searching for MDR inhibitors. Once MDR substrates are identified, the producing plant will be used to isolate MDR inhibitors. A bioassay-driven purification will be used, based on detecting inhibition of cell growth by a test extract in the presence of a sub-inhibitory concentration of an MDR substrate. The structure of newly identified MDR inhibitors will be determined. 3. Characterizing MDR inhibitors. (a) Spectrum of activity regarding the MDR family, and the type of organism will be determined. (b) Interaction of the inhibitor with MDRs will be studied using a transport assay with microbial cells. (c) SAR of MDR inhibitors will be performed. Relation to human health. MDR inhibitors are a new type of plant defense compounds that we are exploring. By potentiating other antimicrobials, MDR inhibitors may provide the key for developing plant antimicrobials into new antibiotics. MDR inhibitors will potentiate the action of conventional antibiotics, aiding eradication of multidrug resistant human pathogens.

This action supports an ultra-high resolution scanning electron microscope (SEM) for research, education, and training in nanoscience and biotechnology. Twenty faculty from Physics, Chemistry, Biology, Pharmacy, Electrical and Computer Engineering, and Mechanical, Manufacturing and Mechanical Engineering will use the instrument to carry out research on several projects. Research areas include bacterial biofilms, microbial diversity in marine environments, protozoan biochemistry and metabolism, nanostructures in spintronic materials, collective electrodynamics of nanostructured oxides and borides, metal-insulator transition in two-dimensional semiconductors, novel green routes to the synthesis of conducting polymers, chemical vapor deposition of metals, and structure-property relations of thin films for electronic applications.

The SEM will facilitate research programs that are supported by awards from several Federal agencies: NSF, AFOSR, ONR, DOE, NIH, as well as from industrial companies and private foundations. The instrument will catalyze new interdisciplinary collaborations in nanoscience and biotechnology. The SEM will be integrated into interdisciplinary graduate courses in nanotechnology and biotechnology offered by several departments. It will be used in undergraduate laboratory courses, undergraduate projects and co-op internships. The SEM will be integrated into the "Building Bridges" and the ACS Project SEED outreach programs for middle and high school students, and the Connections. Program for women and underrepresented populations.

DESCRIPTION (provided by applicant): Our long-term goal is to exploit "unculturable" soil microorganisms for production of broad-spectrum antibiotics against potential biowarfare agents. Soil microorganisms have served as the main source of antibiotics, but only 1% grow in vitro, and have been over mined. In this exploratory project, we will develop approaches to culture the bulk of this previously inaccessible biodiversity, based on a method we developed for growing uncultured marine organisms (Kaeberlein, T., Lewis, K., and Epstein, S.S. (2002) Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment. Science 296:1127-1129). We also aim to achieve proof-of-principle for obtaining antibiotics from uncultured soil microorganisms. The specific aims are: 1. Growing uncultured microorganisms. Methods for growing soil microorganisms in situ will be optimized. This will form the basis for then developing in vitro culturing methods, based on our findings with marine organisms. The two approaches we will use are "domestication", sequential subculture in a diffusion chamber and subsequent adaptation to growth in vitro; and co-culture with symbiotic organisms+ 2. Screening for antimicrobial activity against BW agents. Extracts from individual microorganisms will be obtained and tested for growth inhibition activity with an avirulent Bacillus anthracis* and LVG strain of Francisella tularensis. Hits will be verified against virulent strains of B. anthracis and F. tularensis (NIAID category A). 3. Purification and identification of antimicrobial compounds. Early-stage dereplication will indicate extracts containing antimicrobial compounds of chemical novelty, and these will be used to isolate a pure substance. A sufficiently pure compound will be used to determine MIC with the pathogen strains, and those with high potency will be studied further. Determination of chemical structure will be performed by a combination of MS and NMR methods.

DESCRIPTION (provided by applicant): Our long-term goal is to discover broad-spectrum antibiotics acting against potential biowarfare agents. The goal of this exploratory project is to develop a comprehensive approach to bypass the existing obstacles to drug discovery, which will include delivering active compounds into the pathogen, identifying new classes of antimicrobials, and efficient evaluation of toxicity/efficacy. Previous research in the Lewis laboratory showed that plants synthesize inhibitors of multi-drug resistant efflux pumps that can facilitate delivery of antimicrobials into microbial pathogens. Independently, the Ausubel laboratory developed a pathogenesis model that involves the killing of the nematode worm Caenorhabditis elegans by human microbial pathogens. The nematode can therefore be used as an animal model for primary screening of antimicrobials. This proposal describes experiments designed to merge the complementary technologies developed in these two laboratories to produce a novel approach to antimicrobial drug discovery. The Specific Aims are: 1. High throughput whole-animal screen for novel antimicrobials will be developed using C. elegans infected with a variety of NIAID group A and B agents. These will include diarrheagenic E. coli, S. enterica, and a model gram-positive pathogen E. faecalis. We will also establish whether C. elegans is infected with B. anthracis and F. tularensis. The rationale of the antimicrobial assay is to monitor curing of worms infected with human pathogens, by test compounds. A liquid assay using GFP-labeled C. elegans will be developed into a high-throughput automated assay. 2. Screening for MDR inhibitors and new antimicrobials will be performed in vitro, and in vivo with infected C. elegans. A commercial synthetic compound library, and the NCI collection of extracts will be screened. Comparison of the in vitro and in vivo screens will identify possible compounds that only act in vivo (prodrugs, compounds targeting virulence or other components necessary for in vivo survival, and stimulators of innate immunity). Preliminary results show that the NCI library has hits for both direct and MDR inhibitory activity against all pathogens tested. We will focus on obtaining novel antimicrobials and MDR inhibitors acting against gram-negative pathogens. A combination of such an MDR inhibitor with an antimicrobial compound will produce a broad-spectrum antibiotic. We plan to screen 30,000 compounds/extracts in this pilot study. 3. We will purify and identify antimicrobial compounds. Active extracts will be used to isolate a pure substance. A sufficiently pure compound will be used to determine MIC with the pathogen panel, and those with high potency will be studied further. Determination of chemical structure will be performed by a combination of MS and NMR methods.

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the mechanism of biofilm tolerance to antibiotics. Our preliminary studies suggest that persister cells may be largely responsible for resistance of biofilms and stationary planktonic populations to killing by cidal antimicrobials. The main goal of this proposal is to identify genes responsible for the persister phenotype. This will enable us to directly test the persister hypothesis of biofilm resistance which promises to solve this long-standing riddle, and will provide a new paradigm for the understanding and treatment of biofilm infections. We will use a number of complementary approaches to identify persister genes. We were able to isolate persisters from a high-persistence (hip) strain of E. coli by lysing the bulk of cells with ampicillin, and obtained a preliminary gene expression profile. A detailed time-dependent gene profile of ampicillin treatment will be obtained, providing data for a comprehensive cluster analysis that will indicate candidate persister genes. We will isolate naive persisters using cell sorting with GFP linked to genes that are likely to be expressed in these cells. Additionally, using DNA arrays, we will identify an overlapping set of genes differentially expressed in cells treated with unrelated antibiotics. Persister genes are expected to be among those affecting death and survival. In an independent approach, persister genes will be identified by selection for increased tolerance from a recombinant genomic library. Candidate genes from these approaches will be tested in uniformly constructed strains, each carrying a deletion; and overexpressing the gene from a controllable promoter. Tests with a set of antibiotics will indicate genes that affect persister production in planktonic cultures. Biofilms will then be prepared from persister-deficient or overproducing strains, and tested for tolerance with cidal antibiotics. Correlation between persister status of a strain and biofilm tolerance will provide a definitive test for the persister hypothesis. Identified persister genes will then enable a study of their mechanism of action, which will begin with obtaining an expression profile from strains deficient in; and overproducing the protein of interest. These studies will form the basis for understanding biofilm infections and developing drugs that target persister proteins.

The long-term goal of this project is to discover novel types of broad-spectrum antibiotics capable of effectively eradicating a wide range of potential biowarfare agents, as well as conventional pathogens. In this phase of the project, we will develop a high-throughput screen for a targeted discovery of prodrug antibiotics. The screen is aimed at solving the following problems that have impeded antimicrobial drug discovery: automatically eliminate a high background of generally- toxic molecules in compound library screening; obtain lead compounds with good penetration into Gram negative species, indicative of a broad spectrum of activity; and an ability to sterilize an infection by eliminating persister cells invulnerable to all current antibiotics. Prodrugs such as metronidazole convert inside the cell into a reactive molecule that hits multiple targets. In this regard, an activated prodrug acts like an antiseptic. Our preliminary data show that prodrugs can potentially completely sterilize a stationary state population of E.co//containing persisters. An ability to sterilize an infection can be critically important for treating immunocompromised individuals and multidrug tolerant biofilms that cause 65% of all infectious diseases in developed countries. An activated prodrug will bind covalently to its targets, creating an irreversible sink which will allow it to avoid efflux by MDR pumps of Gram negative species which is the main obstacle in developing broad-spectrum antibiotics. We will use E.'coli,.including the O157:H7 enterohemorrhagic strain that is a Category B waterborne agent in screen development. The process will include test strainconstruction- screening at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases facility in Boston and at an NIH Screening Center, and validation of hits to indicate their prodrug nature. This research will lead to the development of new antibiotics with a broad spectrum of action that will be effective against both bioweapons agents such as Y.pestis, F. tularensis and B. anthracis, and conventional pathogens. The uniquely novel property of these compounds will be their ability to completely sterilize an infection, preventing relapse of a disease and enabling effective treatment of immunodeficient individuals, including the elderly and the very young. PERFORMANCE,SITE(S) (organization, city, state) , Northeastern Mniversity, Boston, MA National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases, Boston, MA PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Lewis, Kim KEY PERSONNEL. See instructions. Usecontinuation pages as neededto provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first; Name eRA Commons User Name Organization Role on Project Kim Lewis k.lewis@neu.edu Northeastern University PI Gabriele Casadei Northeastern University PostdAssociate OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Richard E. Lee Univ of Memphis, TN Consultant Human Embryonic Stem Cells ^ No Q Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from thefollowing list: http://stemcells.nih.qov/reqistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure PermissionStatement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. I I Yes I I No PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 45. Principal Investigator/Program Director (Last, First, Middle): Lewis, Kim The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular basis of bacterial unculturability. ~99% of all bacteria do not grow on synthetic media in vitro, which seriously undermines progress in microbiology, including critical areas related to human health. The established paradigm holds that unculturable organisms are either extremely slow growers, or require some unknown nutrients. In this proposal, we will test an alternative signaling hypothesis of unculturability - most microorganisms evolved to grow in a familiar environment, and will only propagate in response to signals from their surroundings. We have previously developed a method to grow unculturable bacteria by culturing them in a diffusion chamber placed in their natural environment (Kaeberlein, T., Lewis, K., and Epstein, S.S. 2002. Isolating "uncultivable" microorganisms in pure culture using a simulated natural environment. Science 296:1127-1129). Our preliminary findings indicate that growth on synthetic media of isolates from the chamber can be achieved by selection for domesticated variants; or in co-culture with "helper" species. These preliminary observations are consistent with the signaling hypothesis and form the basis of the present proposal. The goal of this project is to discover the genes controlling culturability of a model microorganism MSC33 related to culturable Psychrobacter. We were able to isolate a "domesticated" variant of MSC33 that grows well on a variety of synthetic media. This model will enable us to identify the genetic differences between the parent unculturable and the culturable derivative organisms. This work will be facilitated by a shuttle vector we developed based on an endogenous plasmid from MSC33. Finding the first culturability genes is the essential step toward elucidating the signal transduction pathway controlling growth which we will undertake in a subsequent RO1 project. The reasons preventing the majority of microorganisms from growing in the lab are unknown, and microbiology as a discipline has been restricted to the study of 1% of bacterial species. This project will advance our understanding of uncultivable bacteria, and will help develop tools to grow them. Unculturable bacteria are an enormous untapped source of potentially useful pharmaceutical compounds. Unculturable bacteria also make up most of the human oral and intestinal microflora. This project will advance our understanding of uncultivable bacteria, and will help develop tools to grow them. Unculturable bacteria are an enormous untapped source of potentially useful pharmaceutical compounds. Unculturable bacteria also make up most of the human oral and intestinal microflora. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the mechanism of formation of dormant persister cells exhibiting multidrug tolerance, and to develop a therapy for their eradication. Persisters play an important role in drug tolerance of biofilms. The goal of this project is to obtain an essentially complete set of persister genes and to identify essential targets for future development of anti-persister drugs. We have made significant progress in understanding the nature of persisters and reported the first genes involved in persistence, but a comprehensive set of persister genes remains to be discovered. We have also developed two methods to isolate persisters, which lead to the first persister transcriptome, but a robust, rapid method for obtaining large quantities of persister cells is lacking. In this project, we will aim to resolve these obstacles which are impeding progress in this important field. We will use two organisms - E. coli; and Y. pestis. This will allow us to identify conserved persister genes. Y. pestis survives in the environment well enough to be able to cause an airborne infection. It is possible that the analog of spores - persister cells - aid the survival and spread of the pathogen. This study will lay the background for examining the role of persisters in Y. pestis survival. Based on what we have learned about persisters, it does not appear that any single method will provide a definitive means of identifying persister genes. We therefore propose to use several approaches, and will then synthesize the results of these independent methods to arrive with confidence at a comprehensive set of persister genes. The Specific Aims of this project are: 1. Genomics of high persistence mutants. We will obtain high persistence (hip) mutants with increased production of persisters. Mutated genes will be identified by whole genome sequencing of up to 100 strains in collaboration with a team from The Broad Institute. 2. Isolating persisters and transcription profiling. A. Cell sorting. Advancements in message amplification will allow us to obtain a high-quality transcriptome from small numbers of persisters. We will isolate persisters as described previously, by sorting dim cells from a growing population expressing degradable GFP. A time-dependent transcriptome will point to candidate persister genes. B. Persister capture. Expressing a surface epitope under the control of a persister-specific promoter will allow us to isolate large amounts of persisters. 3. Identification of essential persister maintenance genes. Persister maintenance genes will be identified from screening a large ts mutant library of E. coli. This study will show which functions are critical for persister survival, and will produce attractive targets for developing a dual therapy to eradicate infections. PUBLIC HEALTH RELEVANCE This project is aimed at understanding the nature of bacterial tolerance to antibiotics. Existing antibiotics are unable to eradicate persisters, which are specialized dormant cells present in all bacterial populations. Persisters are largely responsible for many serious relapsing infections. Identifying genes responsible for persister formation will lead to targets for developing an anti-persister therapy. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The increasing incidence of infections by the intestinal anaerobe Clostridium difficile coupled with the spread of drug resistant highly virulent strains dictates the need for developing new therapies for this potentially lethal pathogen. The goal of this project is to develop a novel therapy against C. difficile based on a combination of the well-tolerated antimicrobial berberine and an inhibitor of Multidrug Efflux Pumps (MDRs). Berberine is the principal component of Hydrastis canadensis (Goldenseal), and our previous research determined that its efficacy against bacterial pathogens is limited by MDRs of the MF family. However, MDR inhibitors increase the activity of berberine against gram positive bacteria, including C. difficile, by more than 60 fold. Similar results were found for a conjugate of berberine and an MDR inhibitor. Since the only known mechanism of resistance to berberine is MDR efflux, its combination with an MDR pump inhibitor, either separately or as a hybrid molecule, can result in a powerful antimicrobial. Importantly, the berberine/MDR inhibitor combination had strong bactericidal activity against stationary cells of C. difficile, in contrast to commonly used metronidazole and vancomycin, which were only active against growing bacteria. Upon reaching stationary state, C. difficile produces spores, which are responsible for the relapse of the infection. By killing stationary cells, berberine/MDR inhibitor combination prevents spore formation. This suggests that the combination antimicrobial may have a critical advantage over existing therapeutics by preventing relapse. Inhibitors against MF MDRs are found with high probability in compound libraries (~5%), presenting a unique opportunity to pick and choose leads with attractive properties (including poor absorption). The very high probability of finding MDR inhibitors provides a unique opportunity to rationally manage drug resistance. Conventional antimicrobials eventually fail due to resistance development. The advantage of our approach is that new classes of MDR inhibitors can be relatively easily developed from an enormous base of hit compounds. In this manner, we will be able to stay ahead of pathogen resistance. While finding MDR inhibitor hits has a high probability, developing them into leads presents a challenge as well, since it has been impractical to test a large number of compounds for toxicity and efficacy in vivo. We have recently described a whole-animal screen in C. elegans, that helps overcome these bottlenecks in development. We will use this model to rapidly identify attractive leads which will feed our drug development pipeline. Leads, including those we already identified, will be evaluated in vitro (potency, spectrum, cytotoxicity, absorbance) and then in mice for toxicity, and in a hamster model of C. difficile infection for efficacy. This multidisciplinary project is a collaboration between experts in antimicrobial chemotherapy, medicinal chemistry, C. difficile biology, host-pathogen interactions and veterinary medicine who have worked together on preliminary stages of this project. PUBLIC HEALTH RELEVANCE: Clostridium difficile is the most commonly recognized cause of antibiotic-associated diarrhea (AAD). We will develop an effective therapeutic against drug-resistant C. difficile by disabling the mechanism of resistance to berberine, a widely used antimicrobial from medicinal plants.

DESCRIPTION (provided by applicant): Infectious disease is often untreatable, even when caused by a pathogen that is not resistant to antibiotics. This is the paradox, and the problem that we aim to solve. Microbial populations produce persisters, dormant cells that are not mutants, but phenotypic variants of the wild type that are tolerant to antibiotics. It seemed possible that the presence of persisters could explain the treatment paradox: an antibiotic eliminates most of the population, and once its concentration drops, the surviving persisters start dividing and reestablish the infection. However, prolonged treatment of persisters in vitro with antibiotics which should emulate the in vivo therapy eliminates these dormant cells. The hypothesis. We hypothesize that the agent responsible for untreatable infections is a super-persister cell which carries a high-persister mutation and has induced stress responses. Repeated application of high levels of antibiotics in vitro selects for E. coli hip (high-persister) mutants that have an increased level of persister cells. According to our data, the hip cells are also more drug-tolerant as compared to wild type persisters. We reasoned that periodic application of lethal doses of antibiotics to patients with chronic infections will similarly select for hip mutants. Analysis of longitudinal isolates from a cystic fibrosis patient infected with P. aeruginosa showed that late, but not early isolates are indeed hip mutants. It seems possible that therapy with repeated doses of antibiotic selects hip mutants in many if not all pathogens, and it is these presently overlooked tolerant (rather than resistant) mutants that are ultimately responsible for morbidity of the disease and for the death of a patient. Apart from hip mutations, there seems to be another overlooked, but potentially critical component contributing to tolerance - stress responses. So far, we have known of two seemingly opposite strategies of cell survival - dormancy, which shuts down functions and creates persister cells; and induction of stress responses (heat shock, DNA repair, oxidation stress, etc.) that actively protect the cell from noxious conditions. We propose that these two strategies actually complement each other. If a persister is formed in a population that had expressed stress proteins, then it will shut down antibiotic targets, while retaining protective proteins which will help it survive. In the body, a pathogen is exposes to oxidants, DNA damaging agents, membrane acting agents, and it seems that expression of several stress responses is a norm. The ultimate survivor is then a persister carrying a hip mutation which is formed in a population expressing stress responses. It is this super-persister that is probably responsible for much of untreatable disease and will be the focus of our investigation. The experimental plan will address the following interrelated questions: are hip mutants an important part of chronic infection? Are there super-persisters that combine hip mutations with expression of stress responses? Is tolerance, similarly to resistance, a transmissible trait?

DESCRIPTION (provided by applicant): The majority of gut microbes remain uncultivatable, and this significant obstacle must be overcome to understand the role of the microbiome in human health. The goal of this project is to develop a high-throughput method to grow previously uncultivatable bacteria. Our previous work with uncultivatable microorganisms from the external environment has lead to a number of advances: (1) it is possible to cultivate a substantial number of otherwise uncultivatable bacteria by growing them in situ. When microorganisms are placed into a diffusion chamber which is then returned to their natural environment, a substantial proportion of otherwise uncultivatable microorganisms will grow;(2) reinoculation from chamber to chamber produces domesticated variants that can grow on synthetic media in vitro;(3) many uncultivatable species will grow on synthetic media in co-culture with a cultivable organism from the same environment;(4) we recently discovered the first growth promoting factors for uncultivatable bacteria. An assay-driven purification lead to the identification of siderophores as essential factors produced by helper organisms that trigger growth of uncultivatable bacteria from marine sediment. We find that growth co-culture can be used to obtain uncultivatable organisms from the gut flora. In this project, we will develop a high-throughput approach to co-culture in order to obtain a large collection of previously uncultivatable microorganisms from the gut microbiome. A panel of 24 cultivable gut species representing the main taxonomic groups will be arrayed in a microtiter plate and a platform carrying inserts with a 0.2 5m pore membrane will be placed in the wells. In this manner, each well will be separated into a bottom section inoculated with a given cultivable species and a top section connected with it through pores of the membrane. A suspension from a human fecal sample will then be separated by a cell sorter, and individual cells will be deposited in the upper chamber of each well. After incubation, material from both parts of a well will be collected and tested for growth of the two organisms separately and in co-culture. This will lead to the isolation of uncultivatable species and their helpers. 16S rRNA gene sequence determination will then identify these microorganisms. Whole genome sequencing will be performed for at least ten of the uncultivatable isolates from a variety of taxonomic groups. The genome sequencing will provide an ultimate validation of the proposed approach to obtain novel uncultivatable species from the microbiome. Growth factors will be isolated from the supernatant of corresponding helper organisms by bioassay-guided purification. Structures of the new compounds will be determined. The growth factors will then be examined, individually and in combination, for their ability to enable in vitro cultivation of uncultivatable microorganisms. The tools and approaches we develop are likely to lead to the cultivation of many gut bacteria, and will help us understand the role of the gut microbiome in health and disease. PUBLIC HEALTH RELEVANCE: The majority of gut bacteria are uncultivatable, and do not grow under laboratory conditions for unknown reasons. We find that many of these organisms depend on neighboring, cultivable species for growth. In this project, we will develop a method for large-scale isolation of previously uncultivatable microorganisms by pairing them with the correct helper species, which will enable their genome sequencing, and detailed study of their role in health and disease.

DESCRIPTION (provided by applicant): The slow pace of antibiotic discovery is being outmatched by rapid acquisition of resistance by our pathogens. The need is especially acute in the case of M. tuberculosis, where strains of XDR-TB resistant to the majority of standard therapeutics are rapidly spreading. In this project, we will combine two innovations to develop a drug discovery platform for identifying leads that will be subsequently developed into therapeutics for treating tuberculosis: the use of uncultured bacteria as a unique source of novel antimicrobials;and a high-throughput screen for specific anti-Mtb compounds. Most antibiotics in use today are natural products or their derivatives, obtained from screening soil microorganisms. The main practical problem with discovery from culturable microorganisms is the enormous background of known compounds. At the same time, the vast majority of bacteria, 99% of species, do not readily grow in vitro and are known as uncultured. Our group developed a general method to grow uncultured bacteria by cultivating them in situ. An environmental sample such as soil is mixed with agar and sandwiched between two semi-permeable membranes of a diffusion chamber which is returned to the environment. Isolated colonies of diverse organisms grow in the chamber, and subsequent reinoculation to new chambers produces "domesticated" variants capable of growing on regular Petri dishes in vitro. Our preliminary findings show that this is an excellent source of novel antimicrobials. However, even with this previously inaccessible resource most of the chemistry effort is still wasted on rediscovery of known compounds. We reason that the problem can be resolved if discovery is focused on a species-specific compound. In case of M. tuberculosis, several synthetic compounds have been discovered that specifically act against this organism - INH, ethionamide and pyrazinamide. Natural compounds specifically acting against M. tuberculosis have not been described so far. This means that a screen for specific anti-M. tuberculosis compounds will produce hits that will have a high probability of being novel substances. A specific screen will then largely replace the laborious dereplication. The rationale is to screen in parallel against M. tuberculosis and a different organism, S. aureus. A pilot screen showed an excellent specific hit rate of 1.5% for extracts from uncultured species acting against M. tuberculosis. This screen will be optimized in the proposed project. In order to properly validate the screen, we will need to demonstrate that it is indeed capable of identifying novel compounds acting specifically against M. tuberculosis. We will therefore dereplicate the hits, the structure of unknowns will be determined, and their mode of action will be established. Once developed and validated in this project, the screen will be used in an HTS format for large-scale drug discovery. A combination of a unique, untapped source - uncultured bacteria - and a specific screen is likely to lead to novel compounds to combat drug-resistant M. tuberculosis. PUBLIC HEALTH RELEVANCE: In this project, we will establish a method that allows to rapidly identify antimicrobial compounds for developing drugs to treat tuberculosis. The method is based on using a unique source of antimicrobial compounds - bacteria that do not normally grow in the lab and are known as "unculturable". We will also look for compounds that act specifically against the pathogen M. tuberculosis, which will avoid killing of the good bacteria of our gut flora.

Abstract Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the US. When treated early, the disease is usually easily treated with short courses of antibiotics. However, if allowed to progress to late stage symptoms such as arthritis or encephalopathy, longer courses of up to 28 days of antibiotics are recommended. Even with longer courses of antibiotics, a significant proportion of patients with Lyme arthritis will not improve and will require additional courses of treatment. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In a preliminary study, we find that B. burgdorferi forms drug-tolerant persister cells similar to those formed by other pathogens. In a pilot Tn-seq experiment, we identified candidate genes involved in persister formation in B. burgdorferi treated with ceftriaxone. The goal of this project is to identify the mechanisms of persister formation in Borrelia burgdorferi, which will help understand their role in drug tolerance. Studying E. coli as a model organism, we identified toxin/antitoxin (TA) modules as a major contributor to formation of dormant persister cells. However, B. burgdorferi lacks TA modules, suggesting that it evolved independent mechanisms of drug tolerance. We developed several complimentary methods to identify persister genes, and these will be applied to the study of B. burgdorferi. First, we will expand our initial identification of mutations that affect persister formation using Tn-seq with additional clinically relevant antibiotics. Transcriptome analysis will be performed on persisters isolated by lysing the culture with a cell wall acting antibiotic; and by sorting persisters following a metabolic marker. High-persister mutants (hip) will be obtained by selection in vitro, and the mutant genes identified by whole genome sequencing. We will also screen for hip mutants from wild ticks and patients to understand if these could account for variations in disease manifestation and response to antibiotics. Priority will be given to candidates that are identified by different approaches. Opposite effects of deletion vs. overexpression of a gene of interest on drug tolerance will validate a persister gene. A detailed examination of the genes will lead to a comprehensive understanding of the mechanism of persister formation and drug tolerance in this pathogen, informing development of better treatments.

Abstract Most antibiotics resulted from the Waxman platform, screening of soil microorganisms, but this limited resource was overmined by the late 60s. In the absence of a platform, compounds are introduced slower than pathogens acquire resistance, and the result is a human health crisis. The recent President's executive order ?Combating Antibiotic-Resistant Bacteria? underscores the significance of this problem. In this Program, we will develop an effective discovery program based on exploiting uncultured bacteria to resolve the bottleneck of antimicrobial drug discovery. Uncultured bacteria are an untapped source of secondary metabolites, and we developed methods to grow them and mine for antibiotic discovery. We discovered 25 new compounds from this source so far, including lassomycin, a novel compound with specific activity against the ClpP1P2C1 protease of M. tuberculosis; and teixobactin, a novel inhibitor of peptidoglycan synthesis which is essentially free of resistance development. However, the real potential of uncultured bacteria remains unrealized - the background of knowns and toxic compounds has been the main bottleneck even for this untapped source of chemical diversity. We propose to solve this problem by introducing transcriptome analysis as a rapid tool to identify promising compounds from uncultured bacteria. Compounds affecting the same target produce distinct transcription profiles that cluster together. This approach allows us to classify compounds as known; novel hitting a known target; novel hitting a new valuable target; hitting an undesirable target; or a nuisance compound lacking specificity. In a pilot study, we determined that crude extracts from producing strains can be used to generate transcriptome profiles in a test organism to identify targets, and deduce the presence of a potentially valuable compound. In the proposed project, we will create a database of transcription profiles from known antimicrobials, develop effective computational tools for transcriptome analysis, and will interrogate transcriptomes from a large number of extracts and their fractions from uncultured bacteria. Lead molecules will be validated in vitro and in an animal efficacy model. The end result of the project will be a novel discovery platform, new targets, and lead compounds for drug development. The project is a collaboration between Kim Lewis, PD/PI (NU), an expert in antimicrobial drug discovery and resistance; Karen Nelson, Co-Investigator (JCVI), an expert in genomics, meta-omics approaches and computational biology; and Amy Spoering, Co- Investigator (NovoBiotic), an expert in drug discovery from uncultured bacteria. These experts collaborated on producing preliminary data for this Program.

Abstract Drug-resistant pathogens are causing a human health crisis. The overall goal of this Program is to resolve the bottleneck in the antimicrobial pipeline by developing an effective platform to discover novel compounds produced by an untapped source of chemical diversity, uncultured bacteria. The goal of this component project is to identify the mode of action of known antimicrobials with unknown target; and compounds produced by uncultured bacteria. Natural products have been all but abandoned by the Industry due to overmining of culturable bacteria and diminishing returns, but surprising discoveries keep coming from this group of compounds, both new and old. We discovered that acyldepsipeptide is capable of killing dormant persisters and sterilizes an incurable biofilm infection activating proteolysis. Novel species-selective compounds have been described ? cyclomarin corrupts the essential ClpP1P2C1 protease of M. tuberculosis and lassomycin, which we recently isolated from an uncultured bacterium, inhibits this protease and simultaneously activates its ATPase, resulting in killing dormant cells. Aspergillomarasmine, a rediscovered old compound, inhibits metal ?-lactamases which are insensitive to available therapies. Finally, we recently discovered teixobactin, a novel inhibitor of peptidoglycan synthesis from Elephtheria terrae, an uncultured ?-proteobacterium. The compound binds to both lipid II and lipid III and is essentially free of resistance. We will undertake a large effort to identify the MOA of 500 known compounds with unknown targets. Their transcriptomes (Project II) will indicate specificity of action, and candidates will be evaluated in this project. Resistant mutants will be obtained, and sequencing of the target will provide independent confirmation. The MOA of a selected number of attractive candidates for development will be studied in more detail. This will considerably enrich our knowledge of what evolution determined to be a good target and facilitate the determination of MOA of new compounds produced by uncultured bacteria. Laborious chemical dereplication is a bottleneck that severely limits the rate of discovery of natural products. Rapid detection of the likely MOA of compounds present in extracts and fractions by transcriptome analysis (Project II) will resolve this bottleneck. Candidate leads from both old known compounds and the ones we discover from uncultured bacteria will be characterized in vitro and in vivo for potency, toxicity, and efficacy. For compounds with bactericidal activity, we will examine in detail activity against dormant persister cells and biofilms. Compounds that show good efficacy will become validated leads for advanced drug development. Unleashing the production potential of uncultured bacteria is likely to tip the balance in the standoff with pathogens in our favor. Determining the MOA of candidate compounds, together with their in vitro and in vivo properties will validate this drug discovery platform. The leads we characterize will become candidates for drug development.

The long-term goal of the Program Project is to develop a platform for the discovery of natural product antimicrobials, and to provide leads for drug development. This platform will resolve the current bottleneck in antimicrobial discovery ? lack of good lead compounds. Three PIs from different Institutions are involved in this ambitious project. The related projects are I) Uncultured bacteria for the discovery of novel antimicrobials; II) Antibiotic dereplication by transcriptome analysis; III) Antimicrobial evaluation and development. The essential innovation of our proposed project is to use rapid dereplication by transcription profiling to identify attractive compounds harbored by uncultured bacteria. The objective of the Administrative core is To promote and facilitate the achievement of the goals of the Program Project. The Program Director will be responsible for the overall program and for overseeing the Administrative core. The major priority of the Program Director (PD/PI) is to promote the progress and success of the three research projects and thereby the success of the Program Project. His efforts will be augmented by a Program Advisory Committee and by the Administrative Core Manager. The Administrative Core Manager will coordinate the day-to-day interactions among the PIs and personnel of the three interrelated projects. The Manager will work with the PD/PI, component project PIs and Executive Committee and the Program Advisory Committee to assist with the scientific, administrative and fiscal oversight. The Manager will facilitate communications throughout the Program, and arrange group meetings and teleconferences for members of the project and for meetings of the Program Advisory Committee. The Manager will handle the administrative details of the Program and, interactions with the NIAID and assist in the preparation of regular reports to the NIAID. The Manager will provide the PD/PI with regular reports on the financial status of each project and organize annual meetings of the Project Leaders to review and make adjustments to project budgets. The Core Manager will work with an IT expert to create and maintain a Program website. The website will enable project participants to collect, store and analyze their data from all of the projects of the center; the website will also serve to disseminate information on the Program.

Abstract The goal of the project is to determine the nature of bacterial drug tolerance. Two different types of mechanisms allow bacteria to evade killing by antibiotics ? resistance; and tolerance conferred by persister cells. Unlike resistance, our knowledge of tolerance is limited. Paradoxically, most pathogens that cause chronic infections recalcitrant to antimicrobial chemotherapy are not drug resistant. Tolerance has been linked to persisters, a small subpopulation of dormant cells that survive antibiotics. Many chronic infections are associated with biofilms, which protect persisters from the immune system. An understanding of the mechanism of persister drug tolerance will close a significant gap in knowledge and will contribute to the development of better approaches to treat chronic infections. The current paradigm, based primarily on the study of E. coli, holds that mechanisms of persister formation are not conserved among bacteria, and are governed by toxin-antitoxin modules (TA). However, we recently reported that in S. aureus, TAs play no role in persister formation. Rather, a stochastic decrease in ATP in rare cells produces dormant persisters. We then found that a decrease in ATP is linked to persister formation in E. coli as well. We also established that while some TAs play a role in persister formation under specific conditions in E. coli, this is not the main mechanism. In this project, we will determine the general mechanism by which persisters form in bacteria using E. coli, a representative Gram negative pathogen, and S. aureus, a Gram positive species,. Our preliminary data indicate that stochastic variation in expression of energy producing components - Krebs cycle and glycolytic enzymes - leads to low ATP and persisters. In this project, we will use direct reporters for protein expression and ATP to establish causality between energy producing components and persisters. Apart from conventional time-lapse microscopy, we will take advantage of the ?mother machine?, a massively parallel microfluidics instrument that allows simultaneous analysis of millions of individual cells. Another important unanswered question is the link between persisters and the clinical manifestation of disease. While indirect evidence points to persisters, causality is yet to be established. In this project, we will design pathogen strains with diminished; and overexpressed production of persisters, and link their levels to antibiotic tolerance in biofilm models of murine chronic infection. This project will provide a new paradigm for the understanding of recalcitrance of chronic diseases, and new tools for the study of persisters. This is a multi-PI collaboration between Dr. Kim Lewis, a microbiologist who pioneered the studies of persisters in chronic infections, and Dr. Johan Paulsson, a biophysicist who pioneered massively parallel single-cell analysis.

The Major Research Instrumentation (MRI) program and the Historically Black Colleges and Universities-Undergraduate Program (HBCU-UP) together with the Office of Multidisciplinary Activities (OMA) in the Mathematical and Physical Sciences (MPS) directorate provide support for the acquisition of a cryogen-free Physical Property Measurement System DynaCool (PPMSD) instrument at Howard University. This acquisition supports Howard University researchers' participation in the National Quantum Initiative and the Materials Genome Initiative. The PPMSD provides a state-of-the-art resource to support students and researchers in cutting-edge quantum and materials science research. The PPMSD enhances the active learning experience at the undergraduate and graduate levels in science and engineering departments. Students receive training in magnetic, electrical transport, and heat capacity measurements, experimental data analysis, scientific writing, and presentation skills, which enhances their competitiveness with prospective employers in academia and industry. This, in turn, attracts the next generation of science and engineering students from underrepresented groups who can acquire the requisite skills and then go on to be leaders in their respective fields.

The PPMSD provides the ability to perform variable temperature and magnetic field dependent magnetic, electrical transport, and heat capacity studies relating to quantum materials, magnetic materials, functional materials, and quantum devices. The PPMSD provides advanced research capabilities to undertake several basic and applied research projects that enhance understanding of: (1) the effects of dimensional crossover in mesoscale spin glass dynamics and the role it plays on cooperative phase transitions; (2) the relationships between magnetic and structural entropies, and the performance of magnetocaloric materials; (3) the relationships between electronic transport and magnetic properties in porphyrin molecular junctions; (4) the effects of adatom doping on magnetic, spectroscopic, and transport properties of two-dimensional materials, such as graphene; (5) magnetic and spin transport properties of single molecular magnets covalently bonded between the two ferromagnetic electrodes of a magnetic tunnel junction; and (6) the study of electrical and magnetic properties of recovered high-pressure phases of novel carbon-based clathrate materials.

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.

The incidence and geographic distribution of Lyme disease in the U.S. has increased steadily since its first description in 1977. Efforts to stem the spread of the disease through controlling the population of its tick vector and/or the mouse reservoirs of the disease have met with only limited success. The only approved human vaccine to protect against Lyme disease was removed from the market by its manufacturer further highlighting the need for new approaches to controlling the disease. In this project, we propose the development of a novel antibiotic targeted towards the mouse and tick reservoirs of the disease. This proposal combines the expertise in drug development of Dr. Kim Lewis? laboratory, the expertise in Borrelia burgdorferi biology in Dr. Linden Hu?s laboratory and the field expertise of Dr. Sam Telford?s laboratory. Treatment of mice with an antibiotic, doxycycline, has been shown to be highly effective in eradicating Borrelia burgdorferi from its reservoir hosts. However, there is legitimate concern for development of resistance, both in B. burgdorferi and in other organisms that may be exposed to the antibiotic should it be widely distributed. Doxycyline is an important antibiotic in the treatment of multiple different human infections and in some cases such as Anaplasma or Rocky Mountain Spotted Fever, the only approved agent available. We have identified an antibiotic, hygromycin A (HygA), that is highly active against B. burgdorferi but has limited activity against other human pathogens. Its mechanism of action is different from other human antibiotics. In our preliminary data, we have shown that it is very effective in clearing B. burgdorferi from infected mice when given orally by gavage or in bait formulations. In this proposal, we will complete the steps in developing HygA as an environmental antibiotic and perform a limited field trial on an isolated island off the coast of MA to test its ability to control infection rates in ticks and mice. In Aim 1, we will establish the pharmacodynamics, stability and safety profile of HygA in Peromyscus mice. We will determine optimum concentrations for bait distribution and perform simulation studies of bait uptake and clearance in caged animals. In Aim 2, we will attempt to induce resistance to HygA in B. burgdorferi and in other organisms of human importance that are likely to encounter HygA in the environment. We will confirm that if HygA resistance develops, it does not cause concomitant resistance to other antibiotics with human applications. Finally, in Aim 3, we will perform a limited field trial on an isolated island that is endemic for B. burgdorferi in ticks and mice to establish the efficacy of a HygA based reservoir targeted antibiotic approach. This study has the potential to have a major impact on human Lyme disease by controlling the organism in its major reservoirs. By utilizing an antibiotic that has a narrow spectrum of activity and does not have human applications, we hope to replicate the success seen with doxycycline, which is arguably the most successful trial of any environmental approach to eradication to date, without the attendant concerns for resistance.

Abstract We are currently experience a crisis of antimicrobial resistance, and the World Health Organization identified the need to combat drug resistant Gram negative pathogens as ?critical?. The last class of compounds acting against Gram negative bacteria was introduced over 50 years ago. The most effective strategy to prevent antimicrobial resistance is by introducing novel compounds that act against AMR pathogens and have low probability of resistance development. We recently identified a novel antimicrobial, teixobactin, that shows no detectable resistance against Gram positive bacteria (Ling et al., Nature 2015). Teixobactin is currently in IND-enabling development against important Gram positive AMR infections such as MRSA bacteremia. We more recently discovered another natural product antibiotic, darobactin, with unusually low resistance development that acts against Gram negative AMR pathogens. While these are encouraging advances, a considerable effort is spent on rediscovery of the same producing species and compounds. We reason that mining the rich diversity of Brazilian soils with our approaches will provide access to novel antibiotic to combat the AMR crisis. The Brazilian rain forest has the highest diversity of plants and animals, and we expect the same to be true for microorganisms. Indeed, microorganism directly or loosely associate with eukaryotes, and degrade their products. The goal of this project is to evaluate soils from Brazil for their microbial diversity, to screen isolates for compounds with Gram negative activity, and to isolate and validate novel antimicrobials. If we are able to find novel antimicrobials from soils of New England, it is reasonable to expect that the pace of discovery will increase once we access an environment with the richest diversity on the planet. This project brings together Dr. Kim Lewis, Northeastern University, an expert in drug resistance and drug discovery, and Dr. Monica Pupo, University Sao Paulo, an expert in natural products chemistry. These scientists collaborated on writing the proposal and will work closely together on its execution.

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).

Howard University is a private, federally chartered historically Black University in Washington, DC with more than 10,000 students pursuing 120 areas of study; and currently classified as a High Research Activity Institution. Materials science and engineering (MSE) related research at Howard focuses primarily on quantum materials, optics and photonics and quantum technologies (computing, communications, and sensing). The research focus of this Partnership for Research and Education in Materials (PREM) with Columbia University Materials Research Science and Engineering Center (MRSEC) is on interaction of light with matter for new optical and electrical devices for the quantum information age. This collaboration, namely Partnership for Research and Education in Superatomic and 2D Materials (PRES2M), is to establish the framework needed to pursue new PREM-Pathway for faculty and students at Howard University in MSE. In particular, the following is envisioned: an integrated research and education program that increases participation of undergraduate students in MSE-related research mentored by graduate student trainees and teacher-scholar postdoctoral associates in MSE to strengthen existing diversity efforts at Columbia and provide a foundation for the MSE degree-granting program at Howard. The PREM pathway focuses on matriculation of undergraduate students in MSE-related disciplines to allow for significant growth in the total number of Black students that attain degrees. Furthermore, the collaboration between Howard and Columbia University MRSEC is aimed at impacting current diversity efforts at Columbia with respect to K-12 outreach, undergraduate student research opportunities, and graduate student admissions.

The primary research goal of PRES2M is a better understanding of the quantum phenomena within functional materials, such as, 2D materials and superatomic molecules through a cyclic interplay between materials synthesis, processing, analysis, and theory. The research is aimed at altering material properties to manipulate light-matter interaction including new experimental techniques for thermal tunablity of ultra-strong coupling in 2D nanopatterned films and plasmon-enhanced conductance. This research builds on the materials science of 2D and superatomic materials and applications of quantum phenomena, currently pursued at the Columbia University MRSEC. The goal of this partnership is to fundamentally impact condensed matter physics and materials science and engineering. Through the MRSEC, Howard faculty and students have access to infrastructure, new materials, and expertise in quantum materials research. With the PRES2M, there is an expanded collaboration between both institutions and additional opportunities for co-mentorship of students and postdoctoral associates, joint projects and publications as well as student/faculty exchanges.

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.