Bio


Assistant Professor Lynette Cegelski's research is inspired by the challenge and importance of elucidating chemical structure and function in biological systems and the need for new and unconventional approaches to solve outstanding problems in biology and medicine. The Cegelski laboratory has developed a unique set of tools, particularly integrating solid-state NMR spectroscopy with biochemistry and microbiology, to determine atomic- and molecular-level detail in macromolecular assemblies, intact cells, and bacterial biofilms. Coupled with small-molecule screening and inhibitor discovery, they are driving the development of new strategies to address the global challenge of antibiotic resistance and infectious disease.

Lynette Cegelski completed her undergraduate studies in Chemistry at SUNY-Binghamton, New York (B.S. summa cum laude and Phi Beta Kappa 1998), where she participated in research to determine the microtubule-bound conformation of the anti-cancer drug Taxol by REDOR solid-state NMR. This formative experience motivated her move to Washington University to conduct her PhD training in the laboratory of Professor Jacob Schaefer, where she trained as a solid-state NMR spectroscopist (Ph.D. Biophysical Chemistry 2004). She investigated cell-wall and whole-cell systems and examined photosynthesis and photorespiration in intact leaf NMR experiments. She gained expertise in Microbiology and Infectious Disease research as a postdoctoral fellow in Molecular Microbiology at the Washington University School of Medicine, working with Professor Scott Hultgren. There, she introduced the first small-molecule inhibitors of functional amyloid assembly in bacteria. She joined the faculty of the Stanford Chemistry Department in 2008. Her work has garnered early career awards, including the Burroughs Wellcome Career Award at the Scientific Interface, the 2010 NIH Director’s New Innovator Award, and the National Science Foundation CAREER Award.

Current research in the Cegelski Lab examines bacterial cell-wall composition and, beyond the cell surface, how bacteria self-assemble extracellular structures and use these as building blocks to generate biofilm architectures. Parallel efforts involve the dissection of modes of action of newly discovered antimicrobials and anti-virulence compounds. Lab members employ biophysical and biochemical tools, develop new assays and protocols, and design new strategies using solid-state NMR spectroscopy to examine assemblies such as amyloid fibers, bacterial cell walls, and biofilms. Recent discoveries have emerged from work with E. coli, S. aureus, Vibrio cholerae, and Pseudomonas aeruginosa. Translationally, small-molecule screening efforts have identified biofilm inhibitors that are being tested as potential inhibitors of pathogenesis in vivo.

The laboratory recently discovered that E. coli produces a chemically modified form of cellulose that contributes to the integrity of the biofilm extracellular matrix. Its presence evaded detection through decades of research on bacterial cellulose due to the challenges associated with using solution-based methods to interrogate biomass breakdown products. The laboratory is pursuing several avenues related to manipulations and applications of this new cellulosic material.

Additional targets of study include functional amyloid fibers termed curli and the mechanism by which bacteria use curli together with cellulose to construct biofilm architectures. The curli system is notable as a dedicated amyloid-assembly machinery and inhibitors of curli assembly may also hold promise as inhibitors of amyloids associated with human disease.

Collectively, the Cegelski Research Program is positioned at the scientific interface of Chemistry, Biology, and Engineering and is revealing new bacterial structures, new anti-infective targets, and inhibitors of bacterial adhesion and biofilm formation.

Academic Appointments


Honors & Awards


  • NSF CAREER Award, National Science Foundation (2015)
  • Hellman Faculty Scholar Award, Hellman Fellows Fund (2012)
  • Terman Fellowship, Stanford University (2011)
  • NIH Director's New Innovator Award, National Institutes of Health (2010)
  • Career Award at the Scientific Interface, Burroughs Wellcome Fund (2008)
  • Terman Fellowship, Stanford University (2008)

Professional Education


  • Postdoc, Washington University School of Medicine, Molecular Microbiology (2008)
  • PhD, Washington University, Chemistry (2004)
  • BS, Binghamton University, SUNY, Chemistry (1998)

Patents


  • Lynette Cegelski, Ji Youn Lim. "United States Patent 9,271,493 Methods for Microbial Biofilm Destruction", Leland Stanford Junior University, Apr 11, 2016

Current Research and Scholarly Interests


Our research program integrates chemistry, biology, and physics to investigate the assembly and function of macromolecular and whole-cell systems. The genomics and proteomics revolutions have been enormously successful in generating crucial "parts lists" for biological systems. Yet, for many fascinating systems, formidable challenges exist in building complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell factories. We are inspired by the need for new and unconventional approaches to solve these outstanding problems and to drive the discovery of new therapeutics for human disease.

Our approach is different from the more conventional protein-structure determinations of structural biology. We employ biophysical and biochemical tools, and are designing new strategies using solid-state NMR spectroscopy to examine assemblies such as amyloid fibers, bacterial cell walls, whole cells, and biofilms. We would like to understand at a molecular and atomic level how bacteria self-assemble extracellular structures, including functional amyloid fibers termed curli, and how bacteria use such building blocks to construct organized biofilm architectures. We also employ a chemical genetics approach to recruit small molecules as tools to interrupt and interrogate the temporal and spatial events during assembly processes and to develop new strategies to prevent and treat infectious diseases. Overall, our approach is multi-pronged and provides training opportunities for students interested in research at the chemistry-biology interface.

2016-17 Courses


Stanford Advisees


Graduate and Fellowship Programs


All Publications


  • Influence of the amyloid dye Congo red on curli, cellulose, and the extracellular matrix in E. coli during growth and matrix purification Influence of the amyloid dye Congo red on curli, cellulose, and the extracellular matrix in E. coli during growth and matrix purification Reichhardt, C., McCrate, O. A., Zhou, X., Lee, J., Thongsonboom, W., Cegelski, L. 2016; 408 (27): 7709-7717
  • Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers PLOS ONE Reichhardt, C., Jacobson, A. N., Maher, M. C., Uang, J., McCrate, O. A., Eckart, M., Cegelski, L. 2015; 10 (10)
  • Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Romaniuk, J. A., Cegelski, L. 2015; 370 (1679)

    Abstract

    The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms.

    View details for DOI 10.1098/rstb.2015.0024

    View details for PubMedID 26370936

  • Cell-Based High-Throughput Screening Identifies Rifapentine as an Inhibitor of Amyloid and Biofilm Formation in Escherichia coli ACS INFECTIOUS DISEASES Maher, M. C., Lim, J. Y., Gunawan, C., Cegelski, L. 2015; 1 (10): 460-468
  • Bottom-up and top-down solid-state NMR approaches for bacterial biofilm matrix composition JOURNAL OF MAGNETIC RESONANCE Cegelski, L. 2015; 253: 91-97

    Abstract

    The genomics and proteomics revolutions have been enormously successful in providing crucial "parts lists" for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The "sum-of-the-parts" bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture.

    View details for DOI 10.1016/j.jmr.2015.01.014

    View details for Web of Science ID 000353184300010

    View details for PubMedID 25797008

  • Spectral Snapshots of Bacterial Cell-Wall Composition and the Influence of Antibiotics by Whole-Cell NMR BIOPHYSICAL JOURNAL Nygaard, R., Romaniuk, J. A., Rice, D. M., Cegelski, L. 2015; 108 (6): 1380-1389

    Abstract

    Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Macromolecular and whole-cell NMR spectroscopy allowed us to observe the full panel of carbon and nitrogen pools in S. aureus cell walls and intact whole cells. We discovered that one-dimensional (13)C and (15)N NMR spectra, together with spectroscopic selections based on dipolar couplings as well as two-dimensional spin-diffusion measurements, revealed the dramatic compositional differences between intact cells and cell walls and allowed the identification of cell-wall signatures in whole-cell samples. Furthermore, the whole-cell NMR approach exhibited the sensitivity to detect distinct compositional changes due to treatment with the antibiotics fosfomycin (a cell-wall biosynthesis inhibitor) and chloramphenicol (a protein synthesis inhibitor). Whole cells treated with fosfomycin exhibited decreased peptidoglycan contributions while those treated with chloramphenicol contained a higher percentage of peptidoglycan as cytoplasmic protein content was reduced. Thus, general antibiotic modes of action can be identified by profiling the total carbon pools in intact whole cells.

    View details for DOI 10.1016/j.bpj.2015.01.037

    View details for Web of Science ID 000351774800011

    View details for PubMedID 25809251

  • Sum of the Parts: Composition and Architecture of the Bacterial Extracellular Matrix JOURNAL OF MOLECULAR BIOLOGY McCrate, O. A., Zhou, X., Reichhardt, C., Cegelski, L. 2013; 425 (22): 4286-4294

    Abstract

    Bacterial biofilms are complex multicellular assemblies that exhibit resistance to antibiotics and contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative data are needed to define the molecular composition of bacterial biofilms. Escherichia coli biofilms are known to contain polysaccharides and functional amyloid fibers termed curli, yet accurate determinations of biofilm composition at the molecular level have been elusive. The ability to define the composition of the extracellular matrix (ECM) is crucial for the elucidation of structure-function relationships that will aid the development of chemical strategies to disrupt biofilms. We have developed an approach that integrates non-perturbative preparation of the ECM with electron microscopy, biochemistry, and solid-state NMR spectroscopy to define the chemical composition of the intact and insoluble ECM of a clinically important pathogenic bacterium-uropathogenic E. coli. Our data permitted a sum-of-all-the-parts analysis. Electron microscopy revealed supramolecular shell-like structures that encapsulated single cells and enmeshed the bacterial community. Biochemical and solid-state NMR measurements of the matrix and constitutive parts established that the matrix is composed of two major components, curli and cellulose, each in a quantifiable amount. This approach to quantifying the matrix composition is widely applicable to other organisms and to examining the influence of biofilm inhibitors. Collectively, our NMR spectra and the electron micrographs of the purified ECM inspire us to consider the biofilm matrix not as an undefined slime, but as an assembly of polymers with a defined composition and architecture.

    View details for DOI 10.1016/j.jmb.2013.06.022

    View details for Web of Science ID 000328100500021

    View details for PubMedID 23827139

  • Curcumin as an amyloid- indicator dye in E. Coli CHEMICAL COMMUNICATIONS McCrate, O. A., Zhou, X., Cegelski, L. 2013; 49 (39): 4193-4195

    Abstract

    We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli.

    View details for DOI 10.1039/c2cc37792f

    View details for Web of Science ID 000317931500021

  • REDOR NMR for Drug Discovery Bioorganic and Medicinal Chemistry Letters Cegelski, L. 2013
  • Dimethyl Sulfoxide and Ethanol Elicit Increased Amyloid Biogenesis and Amyloid-Integrated Biofilm Formation in Escherichia coli APPLIED AND ENVIRONMENTAL MICROBIOLOGY Lim, J. Y., May, J. M., Cegelski, L. 2012; 78 (9): 3369-3378

    Abstract

    Escherichia coli directs the assembly of functional amyloid fibers termed "curli" that mediate adhesion and biofilm formation. We discovered that E. coli exhibits a tunable and selective increase in curli protein expression and fiber assembly in response to moderate concentrations of dimethyl sulfoxide (DMSO) and ethanol. Furthermore, the molecular alterations resulted in dramatic functional phenotypes associated with community behavior, including (i) cellular agglutination in broth, (ii) altered colony morphology, and (iii) increased biofilm formation. Solid-state nuclear magnetic resonance (NMR) spectra of intact pellicles formed in the presence of [(13)C(2)]DMSO confirmed that DMSO was not being transformed and utilized directly for metabolism. Collectively, the chemically induced phenotypes emphasize the plasticity of E. coli's response to environmental stimuli to enhance amyloid production and amyloid-integrated biofilm formation. The data also support our developing model of the extracellular matrix as an organized assembly of polymeric components, including amyloid fibers, in which composition relates to bacterial physiology and community function.

    View details for DOI 10.1128/AEM.07743-11

    View details for Web of Science ID 000302807500039

    View details for PubMedID 22389366

  • Fungal biofilm composition and opportunities in drug discovery. Future medicinal chemistry Reichhardt, C., Stevens, D. A., Cegelski, L. 2016; 8 (12): 1455-1468

    Abstract

    Biofilm infections are exceptionally recalcitrant to antimicrobial treatment or clearance by host immune responses. Within biofilms, microbes form adherent multicellular communities that are embedded in an extracellular matrix. Many prescribed antifungal drugs are not effective against biofilm infections owing to several protective factors including poor diffusion of drugs through biofilms as well as specific drug-matrix interactions. Despite the key roles that biofilms play in infections, there is little quantitative information about their composition and structural complexity because of the analytical challenge of studying these dense networks using traditional techniques. Within this review, recent work to elucidate fungal biofilm composition is discussed, with particular attention given to the challenges of annotation and quantification of matrix composition.

    View details for DOI 10.4155/fmc-2016-0049

    View details for PubMedID 27485639

  • Mechanical Behavior of a Bacillus subtilis Pellicle JOURNAL OF PHYSICAL CHEMISTRY B Hollenbeck, E. C., Douarche, C., Allain, J., Roger, P., Regeard, C., Cegelski, L., Fuller, G. G., Raspaud, E. 2016; 120 (26): 6080-6088

    Abstract

    Bacterial biofilms consist of a complex network of biopolymers embedded with microorganisms, and together these components form a physically robust structure that enables bacteria to grow in a protected environment. This structure can help unwanted biofilms persist in situations ranging from chronic infection to the biofouling of industrial equipment, but under certain circumstances it can allow the biofilm to disperse and colonize new niches. Mechanical properties are therefore a key aspect of biofilm life. In light of the recently discovered growth-induced compressive stress present within a biofilm, we studied the mechanical behavior of Bacillus subtilis pellicles, or biofilms at the air-liquid interface, and tracked simultaneously the force response and macroscopic structural changes during elongational deformations. We observed that pellicles behaved viscoelastically in response to small deformations, such that the growth-induced compressive stress was still present, and viscoplastically at large deformations, when the pellicles were under tension. In addition, by using particle imaging velocimetry we found that the pellicle deformations were nonaffine, indicating heterogeneous mechanical properties with the pellicle being more pliable near attachment surfaces. Overall, our results indicate that we must consider not only the viscoelastic but also the viscoplastic and mechanically heterogeneous nature of these structures to understand biofilm dispersal and removal.

    View details for DOI 10.1021/acs.jpcb.6b02074

    View details for Web of Science ID 000379457200033

    View details for PubMedID 27046510

  • Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy EUKARYOTIC CELL Reichhardt, C., Ferreira, J. A., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2015; 14 (11): 1064-1072
  • Toward a Biorelevant Structure of Protein Kinase C Bound Modulators: Design, Synthesis, and Evaluation of Labeled Bryostatin Analogues for Analysis with Rotational Echo Double Resonance NMR Spectroscopy JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Loy, B. A., Lesser, A. B., Staveness, D., Billingsley, K. L., Cegelski, L., Wender, P. A. 2015; 137 (10): 3678-3685

    Abstract

    Protein kinase C (PKC) modulators are currently of great importance in preclinical and clinical studies directed at cancer, immunotherapy, HIV eradication, and Alzheimer's disease. However, the bound conformation of PKC modulators in a membrane environment is not known. Rotational echo double resonance (REDOR) NMR spectroscopy could uniquely address this challenge. However, REDOR NMR requires strategically labeled, high affinity ligands to determine interlabel distances from which the conformation of the bound ligand in the PKC-ligand complex could be identified. Here we report the first computer-guided design and syntheses of three bryostatin analogues strategically labeled for REDOR NMR analysis. Extensive computer analyses of energetically accessible analogue conformations suggested preferred labeling sites for the identification of the PKC-bound conformers. Significantly, three labeled analogues were synthesized, and, as required for REDOR analysis, all proved highly potent with PKC affinities (∼1 nM) on par with bryostatin. These potent and strategically labeled bryostatin analogues are new structural leads and provide the necessary starting point for projected efforts to determine the PKC-bound conformation of such analogues in a membrane environment, as needed to design new PKC modulators and understand PKC-ligand-membrane structure and dynamics.

    View details for DOI 10.1021/jacs.5600886

    View details for Web of Science ID 000351420800039

  • Characterization of the Vibrio cholerae extracellular matrix: a top-down solid-state NMR approach. Biochimica et biophysica acta Reichhardt, C., Fong, J. C., Yildiz, F., Cegelski, L. 2015; 1848 (1): 378-383

    Abstract

    Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae, the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using ¹³C CPMAS and ¹³C{(¹⁵N}, ¹⁵N{³¹P}, and ¹³C{³¹P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, ¹⁵N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems.

    View details for DOI 10.1016/j.bbamem.2014.05.030

    View details for PubMedID 24911407

  • Molecular Determinants of Mechanical Properties of V-cholerae Biofilms at the Air-Liquid Interface BIOPHYSICAL JOURNAL Hollenbeck, E. C., Fong, J. C., Lim, J. Y., Yildiz, F. H., Fuller, G. G., Cegelski, L. 2014; 107 (10): 2245-2252

    Abstract

    Biofilm formation increases both the survival and infectivity of Vibrio cholerae, the causative agent of cholera. V. cholerae is capable of forming biofilms on solid surfaces and at the air-liquid interface, termed pellicles. Known components of the extracellular matrix include the matrix proteins Bap1, RbmA, and RbmC, an exopolysaccharide termed Vibrio polysaccharide, and DNA. In this work, we examined a rugose strain of V. cholerae and its mutants unable to produce matrix proteins by interfacial rheology to compare the evolution of pellicle elasticity in real time to understand the molecular basis of matrix protein contributions to pellicle integrity and elasticity. Together with electron micrographs, visual inspection, and contact angle measurements of the pellicles, we defined distinct contributions of the matrix proteins to pellicle morphology, microscale architecture, and mechanical properties. Furthermore, we discovered that Bap1 is uniquely required for the maintenance of the mechanical strength of the pellicle over time and contributes to the hydrophobicity of the pellicle. Thus, Bap1 presents an important matrix component to target in the prevention and dispersal of V. cholerae biofilms.

    View details for DOI 10.1016/j.bpj.2014.10.015

    View details for Web of Science ID 000347463000002

  • Putative Hydrogen Bond to Tyrosine M208 in Photosynthetic Reaction Centers from Rhodobacter capsulatus Significantly Slows Primary Charge Separation JOURNAL OF PHYSICAL CHEMISTRY B Saggu, M., Carter, B., Zhou, X., Faries, K., Cegelski, L., Holten, D., Boxer, S. G., Kirmaier, C. 2014; 118 (24): 6721-6732

    View details for DOI 10.1021/jp503422c

    View details for Web of Science ID 000337784100040

  • Solid-state NMR for bacterial biofilms MOLECULAR PHYSICS Reichhardt, C., Cegelski, L. 2014; 112 (7): 887-894
  • Community behavior and amyloid-associated phenotypes among a panel of uropathogenic E. coli BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Lim, J. Y., Pinkner, J. S., Cegelski, L. 2014; 443 (2): 345-350

    Abstract

    Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infection and engage in a coordinated genetic and molecular cascade to colonize the urinary tract. Disrupting the assembly and/or function of virulence factors and bacterial biofilms has emerged as an attractive target for the development of new therapeutic strategies to prevent and treat urinary tract infection, particularly in the era of increasing antibiotic resistance among human pathogens. UPEC vary widely in their genetic and molecular phenotypes and more data are needed to understand the features that distinguish isolates as more or less virulent and as more robust biofilm formers or poor biofilm formers. Curli are extracellular functional amyloid fibers produced by E. coli that contribute to pathogenesis and influence the host response during urinary tract infection (UTI). We have examined the production of curli and curli-associated phenotypes including biofilm formation among a specific panel of human clinical UPEC that has been studied extensively in the mouse model of UTI. Motility, curli production, and curli-associated biofilm formation attached to plastic were the most prevalent behaviors, shared by most clinical isolates. We discuss these results in the context on the previously reported behavior and phenotypes of these isolates in the murine cystitis model in vivo.

    View details for DOI 10.1016/j.bbrc.2013.11.026

    View details for Web of Science ID 000331162900001

    View details for PubMedID 24239885

  • REDOR NMR for drug discovery BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Cegelski, L. 2013; 23 (21): 5767-5775

    Abstract

    Rotational-echo double-resonance (REDOR) NMR is a powerful and versatile solid-state NMR measurement that has been recruited to elucidate drug modes of action and to drive the design of new therapeutics. REDOR has been implemented to examine composition, structure, and dynamics in diverse macromolecular and whole-cell systems, including taxol-bound microtubules, enzyme-cofactor-inhibitor ternary complexes, and antibiotic-whole-cell complexes. The REDOR approach involves the integrated design of specific isotopic labeling strategies and the selection of appropriate REDOR experiments. By way of example, this digest illustrates the versatility of the REDOR approach, with an emphasis on the practical considerations of experimental design and data interpretation.

    View details for DOI 10.1016/j.bmcl.2013.08.064

    View details for Web of Science ID 000325483000001

    View details for PubMedID 24035486

  • Disruption of Escherichia coli Amyloid-Integrated Biofilm Formation at the Air-Liquid Interface by a Polysorbate Surfactant LANGMUIR Wu, C., Lim, J. Y., Fuller, G. G., Cegelski, L. 2013; 29 (3): 920-926

    Abstract

    Functional amyloid fibers termed curli contribute to bacterial adhesion and biofilm formation in Escherichia coli . We discovered that the nonionic surfactant Tween 20 inhibits biofilm formation by uropathogenic E. coli at the air-liquid interface, referred to as pellicle formation, and at the solid-liquid interface. At Tween 20 concentrations near and above the critical micelle concentration, the interfacial viscoelastic modulus is reduced to zero as cellular aggregates at the air-liquid interface are locally disconnected and eventually eliminated. Tween 20 does not inhibit the production of curli but prevents curli-integrated film formation. Our results support a model in which the hydrophobic curli fibers associated with bacteria near the air-liquid interface require access to the gas phase to formed strong physical entanglements and to form a network that can support shear stress.

    View details for DOI 10.1021/la304710k

    View details for Web of Science ID 000314082500009

  • Sum of the Parts: Composition and Architecture of the Bacterial Extracellular Matrix Journal of Molecular Biology McCrate, O. A., Zhou, X., Reichhardt, C., Cegelski, L. 2013
  • Nutrient-Dependent Structural Changes in S. aureus Peptidoglycan Revealed by Solid-State NMR Spectroscopy BIOCHEMISTRY Zhou, X., Cegelski, L. 2012; 51 (41): 8143-8153

    Abstract

    The bacterial cell wall is essential to cell survival and is a major target of antibiotics. The main component of the bacterial cell wall is peptidoglycan, a cage-like macromolecule that preserves cellular integrity and maintains cell shape. The insolubility and heterogeneity of peptidoglycan pose a challenge to conventional structural analyses. Here we use solid-state NMR combined with specific isotopic labeling to probe a key structural feature of the Staphylococcus aureus peptidoglycan quantitatively and nondestructively. We observed that both the cell-wall morphology and the peptidoglycan structure are functions of growth stage in S. aureus synthetic medium (SASM). Specifically, S. aureus cells at stationary phase have thicker cell walls with nonuniformly thickened septa compared to cells in exponential phase, and remarkably, 12% (±2%) of the stems in their peptidoglycan do not have pentaglycine bridges attached. Mechanistically, we determined that these observations are triggered by the depletion of glycine in the nutrient medium, which is coincident with the start of the stationary phase, and that the production of the structurally altered peptidoglycan can be prevented by the addition of excess glycine. We also demonstrated that the structural changes primarily arise within newly synthesized peptidoglycan rather than through the modification of previously synthesized peptidoglycan. Collectively, our observations emphasize the plasticity in bacterial cell-wall assembly and the possibility to manipulate peptidoglycan structure with external stimuli.

    View details for DOI 10.1021/bi3012115

    View details for Web of Science ID 000309805100012

    View details for PubMedID 22974326

  • Quantitative Analysis of Amyloid-Integrated Biofilms Formed by Uropathogenic Escherichia coli at the Air-Liquid Interface BIOPHYSICAL JOURNAL Wu, C., Lim, J. Y., Fuller, G. G., Cegelski, L. 2012; 103 (3): 464-471

    Abstract

    Bacterial biofilms are complex multicellular assemblies, characterized by a heterogeneous extracellular polymeric matrix, that have emerged as hallmarks of persistent infectious diseases. New approaches and quantitative data are needed to elucidate the composition and architecture of biofilms, and such data need to be correlated with mechanical and physicochemical properties that relate to function. We performed a panel of interfacial rheological measurements during biofilm formation at the air-liquid interface by the Escherichia coli strain UTI89, which is noted for its importance in studies of urinary tract infection and for its assembly of functional amyloid fibers termed curli. Brewster-angle microscopy and measurements of the surface elasticity (G(s)') and stress-strain response provided sensitive and quantitative parameters that revealed distinct stages during bacterial colonization, aggregation, and eventual formation of a pellicle at the air-liquid interface. Pellicles that formed under conditions that upregulate curli production exhibited an increase in strength and viscoelastic properties as well as a greater ability to recover from stress-strain perturbation. The results suggest that curli, as hydrophobic extracellular amyloid fibers, enhance the strength, viscoelasticity, and resistance to strain of E. coli biofilms formed at the air-liquid interface.

    View details for DOI 10.1016/j.bpj.2012.06.049

    View details for Web of Science ID 000307427700011

    View details for PubMedID 22947862

  • Plant Cell-Wall Cross-Links by REDOR NMR Spectroscopy JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Cegelski, L., O'Connor, R. D., Stueber, D., Singh, M., Poliks, B., Schaefer, J. 2010; 132 (45): 16052-16057

    Abstract

    We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[?-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

    View details for DOI 10.1021/ja104827k

    View details for Web of Science ID 000284202200047

    View details for PubMedID 20964382

  • REDOR Applications in Biology: An Overview Encyclopedia of NMR Toke, O., Cegelski, L. John Wiley & Sons, Ltd. 2010
  • Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation NATURE CHEMICAL BIOLOGY Cegelski, L., Pinkner, J. S., Hammer, N. D., Cusumano, C. K., Hung, C. S., Chorell, E., Aberg, V., Walker, J. N., Seed, P. C., Almqvist, F., Chapman, M. R., Hultgren, S. J. 2009; 5 (12): 913-919

    Abstract

    Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.

    View details for DOI 10.1038/nchembio.242

    View details for Web of Science ID 000271756900014

    View details for PubMedID 19915538

  • Microbial Adhesion Encyclopedia of Microbiology Cegelski, L., Smith, C. L., Hultgren, S. J. Academic Press. 2009; 3: 1-10