Robert A. Nicholas - US grants

DESCRIPTION (Adapted from applicant's abstract): Neisseria gonorrhoeae is the causative agent of gonorrhea, an STD that continues to be a public health problem. Antibiotic resistance in the gonococci, as well as in many other infectious microorganisms, is an increasing problem worldwide. Although penicillin used to be the antibiotic of choice in treating a gonococcal infection, increased resistance to this antibiotic has necessitated its replacement by 3rd generation cephalosporins or fluorinated quinolones for the treatment of infected individuals. Gonococcal resistance to tetracycline has also dramatically risen. Because of the rapid acquisition of resistance to previously efficacious antibiotics, resistance to currently used antibiotics can not be far behind. The resistance of the gonococci to penicillin occurs either through the plasmid-mediated production of penicillinase or chromosomally-mediated alterations in both membrane permeability and penicillin-binding proteins (PBPs). In chromosomally-mediated resistant N. gonorrhoeae (CMRNG), alterations of primary structures of two essential PBPs (PBPs 1 and 2) lead to a decrease in the affinities of these PBPs for penicillin, thereby rendering the organism resistant to that antibiotic. Susceptible gonococci can be transformed in the laboratory to higher levels of resistance using donor DNA from a highly resistant strain. Interestingly, however, it has proven very difficult to transform gonococci from intermediate-level penicillin resistance to a level of resistance equivalent to the donor strain. The highest level of penicillin resistance is correlated with the presence of an altered PBP1, which indicates that the PBP1 gene is involved in the genetic transformation. The investigator has recently cloned the gene encoding gonococcal PBP1, which will allow him to elucidate the mechanisms involved in the acquisition of high level penicillin resistance in N. gonorrhoeae. A five year research plan has been organized around four specific aims. The investigator proposes to clone the gene encoding PBP 1 from penicillin-resistant strains, identify the mutations in PBP1 that lead to the decrease in affinity for penicillin, and investigate the origin of these mutations. The biochemical and functional activities of PBP1 also will be determined. Using the cloned gene, the genetic events underlying the acquisition of high-level penicillin resistance in CMRNG will be investigated. Finally, the investigator will continue efforts to purify and crystallize a soluble form of PBP2 from both susceptible and resistant gonococci, with the long term goal to define the structural changes in PBP2 from resistant strains that result in a lower affinity for penicillin. The three-dimensional structure of a lethal target of beta-lactam antibiotic also may lead to the rational design of new antibiotics based on molecular modeling of the beta- lactam binding site.

Antibiotic resistance in Neisseria gonorrhoeae remains a very important problem. Penicillin and tetracycline, which were once the antibiotics of choice for treatment of gonococcal infections, are no longer used due to the preponderance of strains resistant to these agents. Resistance to currently recommended antibiotics is also increasing. My laboratory is interested in the mechanisms of chromosomally-mediated antibiotic resistance in the gonococcus, especially those that promote high- level resistance and subsequent treatment failure. Intermediate- level chromosomally-mediated resistance to penicillin and tetracycline is due to three resistance loci. These include the penA gene encoding altered forms of penicillin-binding protein 2 (PBP 2), the mtr loci conferring resistance to hydrophobic agents, and the penB gene, which decreases outer membrane permeability. The genes involved in mediating high-level penicillin resistance, however, have been difficult to identify. Our work during the last funding period has identified two resistance genes, ponA and penC, which together mediate high- level penicillin resistance, and a third gene, tetGC, which confers high-level tetracycline resistance. This proposal outlines experiments to clone and characterize the penC and tetGC genes and to elucidate the mechanisms by which they increase resistance. In addition, we propose experiments that follow up on our structure/function studies of the penB gene product, porin IB, to understand how mutations in this protein increase both penicillin and tetracycline resistance. We also propose studies to complete our work on the crystal structure of penicillin- binding protein 2 (PBP 2), an essential penicillin target, and several mutant forms that display a lower affinity for beta- lactam antibiotics. In addition, we will engage in new structural studies of wild-type and mutant forms of porin IB to explicate in molecular detail how mutations in this protein decrease antibiotic permeability. The combination of genetic, biochemical, biophysical, and structural approaches outlined in this proposal will provide important insight into the mechanisms by which this important human pathogen becomes resistant to antibiotics.

? DESCRIPTION (provided by applicant): This Pharmacological Sciences Training Program (PSTP) is dedicated to training outstanding scientists in the pharmacological sciences. A highly productive and well-funded faculty provide a broad diversity of research areas for trainees that builds on our traditional strengths in receptors and signal transduction, cancer and protein kinases, and neuropharmacology with emerging areas in chemical biology, nanotechnology, genomics and proteomics, stem cells, RNA biology, bioinformatics and systems biology. Students apply to the Biological and Biomedical Sciences Program (BBSP), an admissions portal/first year program for 14 degree-granting departments/curricula in the School of Medicine, Biology and the biological division of Chemistry in the College of Liberal Arts & Sciences, and the Divisions of Chemical Biology & Medicinal Chemistry and Molecular Pharmaceutics in the School of Pharmacy. This umbrella program oversees recruitment and training of first- year graduate students in the biomedical sciences. Last year, the BBSP brought in 88 students, 16 of which were underrepresented minorities (18%). Students carry out three research rotations, take basic first year courses, and, at the end of their first year, choose a mentor and a PhD program for their thesis research. Students joining the PSTP choose from 45-core faculty for their dissertation research. A very strong Medical Scientist Training Program (MSTP) also brings in ~8-9 students per year, 1-3 of which join the PSTP per year. The PSTP consists of formal courses, seminar courses focusing on scientific communication skills, and original doctoral research. Basic courses in cell biology or neurobiology, introductory and advanced courses in pharmacology and physiology, and elective specialized courses are required and are taken in the first and second years. A rigorous and intensive grant-writing course develops skills for identifying an important and innovative research question and hypotheses and formulating a strong set of Specific Aims that test these hypotheses. Presentation courses and a student seminar series provide students with many opportunities to hone their research presentation skills and gain confidence in public speaking. Quantitative skills are developed through strong emphasis on biostatistics, biocomputation, and ligand-receptor binding theory and analysis. Individual Development Plans are drafted for all students, and also are used to identify quantitative skills classes germane to the students' research projects. A robust advisory system oversees the thesis research years of students.

[unreadable] DESCRIPTION (provided by applicant): The proper targeting of proteins to either the apical or basolateral surface of polarized airway epithelial cells is critical for normal lung function. An important example is cystic fibrosis, a disease caused in large part by a single amino acid mutation in the CFTR Cl- channel that prevents its transport to the apical surface of airway cells. Other important proteins, such as G protein-coupled P2Y receptors for extracellular nucleotides, are expressed in airway and other polarized epithelial cells and are targeted to different membrane domains of these cells. Thus, the P2Y1 receptor is expressed exclusively at the basolateral surface of polarized epithelial cells, whereas the P2Y2 receptor is expressed at the apical surface. Very little is known about the sorting signals and mechanisms by which G protein-coupled receptors in general, and P2Y receptors in particular, are targeted to their final destination in polarized cells. In this application, we propose to identify the mechanisms by which P2Y1 and P2Y2 receptors are targeted to opposite surfaces of airway and other polarized epithelial cells. We will first establish the routes by which P2Y1 and P2Y2 receptors are delivered to the basolateral and apical domains of polarized cells. In the second aim, we will identify the targeting domains that direct polarized expression of the two receptors by utilizing both confocal microscopy and quantification of cell-surface expression in MDCK cells. We will delimit the regions that are responsible for polarized expression of the receptors, define the amino acids that are most critical, and determine whether the sorting signals direct targeting of non-sorted receptors. Finally, we will identify proteins that interact with the targeting domains of these receptors by yeast 2-hybrid and proteomics approaches. Once identified, we will characterize these proteins to determine if they direct polarized targeting. These experiments will provide important information into the mechanism by which P2Y receptors achieve their steady-state localization in polarized epithelial cells and generate insights into how G protein-coupled receptors in general are targeted to different membrane domains.

DESCRIPTION (provided by applicant): N. gonorrhoeae, the causative agent of gonorrhea, is responsible for -300,000 infections in the U.S. and over 6 million globally. Antibiotics remain the primary treatment for gonorrhea infections, but antibiotic resistance threatens their continued use. Penicillin and tetracycline were once the antibiotics of choice but are no longer effective, and resistance to fluoroquinolones is rapidly increasing. My laboratory focuses on the molecular mechanisms of antibiotic action in the gonococcus, particularly the [unreadable]-lactam antibiotics such as penicillin, and how resistance arises to these drugs, which in N. gonorrhoeae is an unusually complex and multifactorial process that is not completely understood. We have recently uncovered a surprising role in antibiotic influx of the PilQ secretin, one of the proteins in the Type IV pilus complex, which is involved in adhesion and invasion, twitching motility, and DNA transformation. In Specific Aims 1 and 2, we will follow up on this discovery to establish how PilQ and other Type IV pilus proteins promote the entry of antibiotics, and to define the interactions of these proteins with each other in the Type IV pilus complex. In Specific Aim 3, we outline experiments to clone and characterize a recently identified resistance determinant found in high- level penicillin-resistant clinical isolates. This aim will provide new understanding of the mechanisms of chromosomally mediated resistance and novel insight into gonococcal physiology. In Specific Aim 4, we describe a proteomics approach to identify and characterize proteins that bind to penicillin-binding protein 1 (PBP 1), PBP 2, and the lytic transglycosylase MltA, and thus reveal the form and function of the multi-enzyme complexes that synthesize peptidoglycan. Together, these projects utilize genetic, biochemical, biophysical, and proteomics approaches to generate new insights into the mechanisms of antibiotic action in N. gonorrhoeae.

DESCRIPTION (provided by applicant): Our work over the last funding period has focused on characterization of P2Y receptor targeting in polarized cells. These studies established that seven of the eight known subtypes of P2Y receptors display polarized expression in epithelial cells, with four receptors at the basolateral membrane and three receptors at the apical membrane. We have identified and characterized the sorting signals in five of these receptors, which revealed novel mechanisms by which the receptors achieve polarized expression. These findings will be extended to define the physical and structural properties of the sorting signals in the C-terminal tails of P2Y1, P2Y4 , and P2Y14 receptors. The proteins that bind to P2Y receptor sorting signals and direct their polarized targeting are unknown. We will identify these proteins and define their roles in receptor targeting. Lipid microdomains are implicated in localization of proteins in endothelial and epithelial cells and in the regulation of signaling molecules, including P2Y receptors. We will elucidate the role(s) that lipid rafts and caveolae play in the localization and signaling responses of P2Y receptors and delimit the receptor domain(s) involved in these interactions. Successful completion of these projects will provide major new insights into the mechanistic/structural basis of cell targeting and regulation of signaling activities of a therapeutically important class of receptors in the context of a physiologically relevant cell model. The nucleotide-activated P2Y receptors are proven or potential therapeutic targets for treatment of lung diseases, hypertension, and stroke. In epithelial cells, these receptors are targeted to distinct membrane surfaces, where they regulate ion and water transport across the epithelial monolayer. Although cellular mistargeting of proteins is often associated with disease, the cellular mechanisms for proper protein localization are still not well understood. Our studies will provide novel and important information on the cellular mechanisms of P2Y receptor targeting and the regulation of P2Y receptor signaling activity in epithelial cells.

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae is the etiologic agent of the sexually transmitted infection, gonorrhea. Antibiotics are the mainstay in treating infections, but widespread resistance in N. gonorrhoeae, most notably emerging resistance to ceftriaxone, may soon result in strains that are untreatable with current antibiotics. Thus, new antibiotics against novel targets are desperately needed to stem the tide of resistant bacteria that are becoming a major threat to public health. The goal of this proposal is to optimize inhibitors of LpxC, an essential enzyme in the lipid A biosynthetic pathway, for treatment of N. gonorrhoeae infections. Preliminary data demonstrate that LpxC inhibitors are bactericidal for N. gonorrhoeae and are largely unaffected by established resistance mechanisms. Further development of these novel compounds will be achieved by (1) lead optimization of LpxC inhibitors, (2) evaluation of pharmacokinetic and pharmacodynamic properties of lead compounds, and (3) evaluation of antibiotic efficacies in a mouse model of infection. At the completion of this project, we anticipate having one or more LpxC inhibitors with good pharmacokinetic and pharmacodynamic properties that are potent and efficacious against N. gonorrhoeae both in vitro and in vivo. These studies would meet a number of benchmarks required for assembling an investigational new drug application to the FDA for approval of a new class of antibiotics for treatment of N. gonorrhoeae and other Gram-negative infections.

Project Summary/Abstract Chromosomally mediated resistance to ceftriaxone in Neisseria gonorrhoeae is of great concern in public health. With over 350,000 infections reported in the US, and over 78 million infections estimated world-wide, the possibility of losing the most effective antibiotic for treating gonorrhea is such a concern that the CDC has labeled multidrug resistant N. gonorrhoeae an urgent public health threat. N. gonorrhoeae has become resistant to essentially all antibiotics that have been used historically to treat gonococcal infections, including penicillin, tetracycline, ciprofloxacin, and incidences of resistance to the currently used dual therapy antibiotics, ceftriaxone and azithromycin, are rapidly increasing. Despite all of the research on antibiotics and their lethal targets, the actual mechanism by which bacteria are killed by bactericidal antibiotics is still not entirely clear. It has been proposed that bactericidal antibiotics kill Escherichia coli by a common mechanism, and our studies in N. gonorrhoeae are consistent with this hypothesis. By transforming all known resistance determinants from different resistant clinical isolates into multiple recipient strains, we have determined that whereas we can reach the same MICs of bacteriostatic antibiotics as those of the donor strains, the MICs of a broad range of bactericidal antibiotics are consistently 3-4 fold lower in the transformed strains compared to the donors. This pan-resistance suggests that bactericidal antibiotics kill bacteria by a common mechanism, and that resistant clinical isolates have altered this common mechanism to increase resistance to bactericidal antibiotics. This proposal focuses on the genetic analysis of antibiotic resistance. In Specific Aim 1, we will examine the transcriptional profile of PenR and CephR clinical isolates that display pan-resistance to bactericidal antibiotics, both in the absence and in the presence of a breakpoint concentration of a set of bactericidal and bacteriostatic antibiotics. Moreover, because in clinical settings N. gonorrhoeae never interacts with antibiotics in isolation, but within the host, these studies will also be done in the presence of human-derived endocervical epithelial cells. These studies will test the hypothesis that host cells targeted by gonococci impose stresses distinct from that experienced by gonococci in laboratory media during exposure to bactericidal antibiotics. Once this set of genes is identified, their roles in resistance to bactericidal antibiotics will be investigated. In Specific Aim 2, we will utilize transposon mutagenesis with negative selection to identify genes involved in intrinsic resistance to ?-lactam antibiotics, which will identify new targets for future drug development. The proposed studies build on our past successes in defining the mechanisms of chromosomally mediated antibiotic resistance in N. gonorrhoeae, while utilizing new technologies and approaches to identify genes that modulate resistance to antimicrobials. These studies will enable us to fully understand the complex mechanisms involved in mediating gonococcal resistance to ceftriaxone and other bactericidal antibiotics.

PROJECT SUMMARY The increasing rate of infection and spread of antibiotic resistance in Neisseria gonorrhoeae poses an urgent threat to public health. Knowledge of the pathways and genes that support the emergence and spread of antibi- otic resistance is necessary to develop new strategies for surveillance, diagnosis, and treatment. Despite our understanding of the genes and alleles that confer resistance, significant knowledge gaps remain regarding the factors that contribute to the uneven distribution of resistance across the gonococcal species phylogeny. A core issue that remains poorly explored is the impact of resistance determinants on gonococcal fitness: to what extent do resistance determinants impact fitness, and, if they incur a fitness cost, how does the gonococcus adapt and mitigate these costs? In this proposal, we address these gaps through a comprehensive strategy linking experi- mental and computational identification of compensatory mutations with studies of their mechanisms of action. The overall goal of this project is to determine the impact of mutations that increase resistance to the two most clinically relevant antibiotics for treatment of gonorrhea, ciprofloxacin and ceftriaxone, on bacterial fitness. We will achieve this goal through three specific aims. In Aim 1, we will determine the fitness costs of resistance alleles when transformed into susceptible isolates from different niches and with distinct phylogeny and identify compensatory mutations that mitigate these fitness costs through experimental evolution in the female mouse model. We will examine the two most common ciprofloxacin resistance-conferring alleles (gyrAS91F,D95G and parCS87R) in clinical isolates, and four ceftriaxone resistance-conferring alleles (two variants of penA, the lethal target of ceftriaxone, and newly described variants in rpoB and rpoD), and evaluate the dependence of compen- satory pathways on genomic background. In Aim 2, we will leverage our collection of over 7500 gonococcal genomes from clinical isolates for which we have antibiotic-resistance phenotypes and employ population ge- nomics methods to identify potential compensatory mutations and test these in the mouse model. Moreover, we will define the allelic diversity and distribution of candidates identified in Aim 1. In Aim 3, we will determine the mechanism of action of confirmed compensatory mutations arising from the studies in Aims 1 & 2 using an integrative strategy that examines growth and morphology, transcriptomics, metabolomics, and directed studies of biochemical function. We will also build on preliminary data on compensatory mutations in acnB and mleN for ceftriaxone resistance and on the thiamine biosynthesis pathway in gyrA-mediated quinolone resistance. This interdisciplinary project brings together the complementary and non-overlapping expertise of three lead- ing investigators in the biology and genetics of antibiotic resistance in N. gonorrhoeae, linking the mouse model of gonococcal infection (Dr. Jerse), population genomics (Dr. Grad), and biochemical and physiological charac- terization of resistance-related variants (Dr. Nicholas).

The mission of the Pharmacological Sciences Training Program (PSTP) is to train outstanding scientists in the pharmacological sciences, enabling them to transition to any of the varied career opportunities in the U.S. biomedical research enterprise. A highly productive and well-funded faculty provide a diversity of research areas for trainees that builds on our traditional strengths in receptors and signal transduction, cancer and protein kinases, and neuropharmacology with emerging areas in chemical biology, nanotechnology, genomics and proteomics, stem cells, RNA biology, bioinformatics and systems biology. Students apply to the Biological and Biomedical Sciences Program (BBSP), an admissions portal/first year program for 14 degree-granting departments, an umbrella program that oversees recruitment and training of first-year graduate students in the biomedical sciences. The BBSP admits between on average 75-90 students per year, with an increase to 100 students slated for 2019. A significant portion (20-25%) of matriculating students are underrepresented in the biomedical sciences. Students carry out three research rotations, take a required first year course and another course of their choosing, and, at the end of their first year, select a thesis mentor and a degree-granting PhD program for their thesis research. Students joining the PSTP choose from 46-core faculty for their dissertation research. A very strong Medical Scientist Training Program (MSTP) also brings in ~10 students per year, 0-2 of which join the PSTP per year. The PSTP is designed around a complementary set of training tools, including formal lecture-based courses, seminar courses focusing on scientific communication skills, an immersive grant-writing course, and original doctoral research. Our rationale is that by combining these training approaches, we prepare our trainees for future success. Introductory and advanced courses in pharmacology and physiology, and elective specialized courses are required during the first and second years. A rigorous and intensive grant-writing course develops skills for identifying an important, innovative and tractable research question and hypotheses and formulating a strong set of Specific Aims that test these hypotheses. Presentation courses and a student seminar series provide students with many opportunities to hone their research presentation skills and gain confidence in public speaking. Quantitative skills are developed through strong emphasis on biostatistics, biocomputation, and ligand- receptor binding theory and analysis. Rigor and reproducibility are emphasized across all training opportunities. Individual Development Plans are drafted for all students, and also are used to identify quantitative skills classes germane to the students? research projects. A robust advisory system oversees the thesis research years of students. Faculty rely on evidence-based mentoring practices to best help students achieve shared goals. Based on previous years, we anticipate that we will have 9-11 new, training grant-eligible students entering the PSTP program each year. Students will be appointed to the training grant for a maximum duration of two years, during their second and third year of graduate school. The average time-to-degree over the last five years for all PSTP students, including trainees from underrepresented groups, is 5.5 years, and these students had on average 4 publications, a little less than two of which were first-author publications. Twenty-three percent of current PSTP trainees are from underrepresented groups, and three of our current UR trainees were awarded the prestigious HHMI/Gilliam Fellowship for Advanced Study. Our overall goal is to train a diverse cohort of future scientists by equipping them with critical-thinking, computational, and communication skills and providing exposure to myriad career opportunities needed to obtain a position in a scientific career of their choosing.