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 full genome sequences for an increasing number of organisms and in predicting and determining the structures of a steadily increasing number of proteins. In essence, these data provide 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 factories. We are inspired by the need for novel and unconventional approaches to solve these outstanding problems in biology.
We employ biophysical and biochemical tools, develop new assays and protocols, and are designing new strategies using solid-state NMR to examine bacterial amyloid fibers, bacterial cell walls and biofilms, and membrane proteins. We would like to understand at a molecular and atomic level how cells self-assemble fascinating extracellular structures and how bacteria use these building blocks to construct organized biofilm architectures. We are also engaged in identifying small molecules to interfere with these processes and in understanding the modes of action of newly discovered antibiotics and anti-virulence compounds.
 
Biofilms & Bacterial Cell Walls

Bacteria occupy remarkably distinct niches on our planet and the propensity for bacteria to associate with surfaces and with each other in nearly all ecosystems far exceeds the tendency to persist in suspension, living freely in a planktonic state. Bacteria are found attached to rocks and soil particles, corals and ocean sponges. Bacteria symbiotically colonize plants and humans as well as fish and squid, resulting in mutual benefit to both microbe and host. Pathogenic and unwelcome bacteria colonize host tissues, leading to cellular injury and disease. Specific adhesion strategies have evolved in order to facilitate bacterial attachment. Understanding the molecular mechanisms and functional implications of microbial adhesion is crucial to generating complete descriptions of our ecosystems and attempting to control and prevent the unfortunate and often devastating consequences of infectious diseases.

Bacterial adhesion is an initial and crucial event in the process of biofilm formation. Biofilms are complex, organized bacterial assemblies that exhibit reduced sensitivity to conventional antibiotics, host defenses, and external stresses. Biofilms within the host are implicated in serious and persistent infectious diseases including cystic fibrosis, chronic otitis media, and urinary tract infection. In the environment, biofilms can serve as reservoirs for pathogens and are associated with biofouling.

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Drug Discovery

New antibiotics and anti-infective strategies are needed as common pathogens such as S. aureus and Streptococcus species emerge resistant to “drugs of last resort.” Solid-state NMR spectroscopy has contributed tremendously to our understanding of cell wall architecture in S. aureus and the modes of action of potent vancomycin analogues that are effective against methicillin and vancomycin-resistant S. aureus. We remain engaged in efforts using solid-state NMR to dissect drug modes of action to drive the discovery of new therapeutics.

We also have a strong interest in using small molecules in chemical genetic approaches to interrupt and dissect assembly events and cellular processes in bacteria. Exogenously added small molecules can even serve as probes of biofilm architecture and assembly in the way they were used to study S. aureus cell wall biosynthesis. Multiple methods including AFM, fluorescence spectroscopy and microscopy, and solids NMR can provide complementary detail of the complexes. We are designing new assays to identify and develop target compounds to influence curli biogenesis and amyloid formation. Ultimately, some small molecules may translate our efforts from the laboratory into new anti-amyloid and anti-biofilm agents.

 
Protein Assemblies

Microbial amyloid: structure and function 

E. coli and other Gram-negative bacteria assemble extracellular adhesive amyloid fibers termed curli. Curli are among a growing list of functional amyloids that emphasize Nature’s ability to assemble amyloid fibers that exhibit uniquely tailored physiological functions. Curli mediate cell-surface and cell-cell interactions and serve as an adhesive and structural scaffold to promote biofilm assembly and other community behaviors. Curli biogenesis requires specific nucleation-precipitation molecular machinery encoded by the csgBA and csgDEFG operons. In vivopolymerization of the major curli subunit CsgA into β-sheet-rich amyloid fibers requires the nucleator protein, CsgB. CsgG is a membrane protein and CsgE and CsgF are assembly factors required for the stabilization and transport of CsgA and CsgB to the cell surface. Thus E. coli harbors a tractable and genetically tunable model to dissect the molecular and structural basis of amyloid biogenesis in a cellular context. We are working to transform cartoon representations of the curli fiber and membrane-associated machinery into a molecular model using solid-state NMR spectroscopy together with data from other techniques including microscopy, FRET, and mass spectrometry. As we develop new assays to probe curli biogenesis, we are also working to identify and develop strategies to interfere with amyloid assembly.

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Macromolecular and whole-cell NMR 

Solid-state NMR is a prominent technique in our toolbox. It is well suited to provide compositional and atomic-level insights into complex heterogeneous systems such as intact plant leaves and bacterial biofilms that pose a challenge to analysis by traditional methods. The use of solid-state NMR spectroscopy to dissect macrosystems is powerful, versatile, and broadly applicable to diverse systems, but there are no set protocols. We are developing protocols to introduce stable isotope labels selectively in vivo in bacteria, plants, and cellular systems, often manipulating biosynthetic pathways to take up labeled components such as amino acids, carbohydrates, or metabolites. We track these labels by various solid-state NMR detection schemes to understand how they are incorporated or transformed. The selection and development of pulsed NMR schemes depends on the problem that needs to be solved. Our goals are not typically ones to determine total structures, but rather to connect partial local structure with biological function to solve a wide number of outstanding problems and to understand the fundamental chemistry of biological systems.

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