Research

Overview

Our research is aimed at understanding the dynamic process that allows bacteria to integrate and adapt to their environment. The approach we’ve taken to this problem is to better understand the tools that microorganisms use to interact with themselves and their surroundings. This has led us to pursue extracellular organelle biology with special emphasis on curli fiber biogenesis by E. coli. During the course of our studies, we found that curli share distinguishing features with the medically important pathogenic amyloid fibers that are the hallmark of many neurodegenerative diseases such as Alzheimer’s, Huntington’s, systemic amyloidosis and the prion diseases.

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Freeze-fracture, deep-etch electron micrograph showing colonies of curliated E. coli after 48 hours of growth. Curli fibers surround and connect individual bacteria into small groups.

Protein Misfolding Done Right

The discovery of a natural amyloid protein in E. coli opens up the possibility of performing genetic and biochemical experiments that are impractical or impossible in other amyloid model systems. Our work focuses on three related questions: how curli are assembled on the cell surface, how curli modify community behavior in bacteria, and how curli expression affects the host-pathogen interaction during an infection. We blend microscopy, biochemistry, and genetics in a concerted effort to delineate the structural, functional, and molecular details of curli, a distinct class of bacterial fibers that share properties with amyloid proteins. The curli biogenesis apparatus is possibly the world’s most tractable model system for understanding amyloid formation. But more than that, teasing apart the details of curli biogenesis will give insights into such fundamental bacterial processes as gene regulation, protein secretion and protein folding.

The Facts of Amyloid– Schematic representation of amyloid polymerization. (A) Typical ThT fluorescence kinetics of soluble purified CsgA monomers polymerizing into curli. The lag, growth, and stationary phases are indicated. The blue arrow indicates the end of the lag phase and when amyloid fiber formation initiates. Scale bar 500 nm (B) The transition from soluble, monomeric proteins into polymeric and insoluble amyloid fibers is characterized by distinguishable steps that results in loss or gain of function properties for the protein. In the lag phase, soluble protein assembles into a common intermediate or nucleus that is proposed to be toxic to membranes. The formation of the intermediate is proposed to be the rate-limiting step of amyloidogenesis. Once the nucleus is formed, monomers are templated into growing amyloid fibers causing an increase in ThT fluorescence. When the monomer population is depleted, elongation stops and enters the stationary phase. The green boxes below the schematic highlight properties of some of the functional amyloids, while the red boxes highlight some general properties of disease-associated amyloids.

Curli Biogenesis

Much of the Chapman lab is interested in figuring out how curli amyloid fibers are assembled on the cell surface. Aggregation-prone curli subunits must be chaperoned from the inside of the cell to the outside, where they polymerize into stable amyloid fibers.

Model of curli biogenesis Excluding CsgD, the master curli regulator, all Csg proteins have Sec-dependent signal sequences allowing their secretion into the periplasm. The lipoprotein CsgG forms a pore like structure in the outer membrane. The major subunit protein CsgA and the nucleator CsgB are secreted to the cell surface in a CsgG and CsgE-dependent fashion. CsgF associates with the outer membrane and is required for cell association of the minor curli fiber subunit CsgB. Situated at the cell surface, CsgB nucleates soluble, unstructured CsgA into a highly ordered amyloid fiber. Curli production can be visualized by Congo red binding which is absent in a csgA mutant and by transmission electron microscopy (left inserts). Also shown are two CsgA subunits interacting in a cross-β conformation with the R1-R5 interaction depicted.

Model of curli biogenesis Excluding CsgD, the master curli regulator, all Csg proteins have Sec-dependent signal sequences allowing their secretion into the periplasm. The lipoprotein CsgG forms a pore like structure in the outer membrane. The major subunit protein CsgA and the nucleator CsgB are secreted to the cell surface in a CsgG and CsgE-dependent fashion. CsgF associates with the outer membrane and is required for cell association of the minor curli fiber subunit CsgB. Situated at the cell surface, CsgB nucleates soluble, unstructured CsgA into a highly ordered amyloid fiber. Curli production can be visualized by Congo red binding which is absent in a csgA mutant and by transmission electron microscopy (left inserts). Also shown are two CsgA subunits interacting in a cross-β conformation with the R1-R5 interaction depicted.

Protein and Chemical Chaperones

 

Biology of the Bacterial Extracellular Matrix

Cells in a biofilm are surrounded by an extracellular matrix that protects them from various stresses ranging from antibiotics to the host immune response. Resistance conferred by the extracellular matrix makes biofilm infections difficult to eradicate. Our lab is actively investigating the molecular pathways that control the extracellular matrix components, along with the role of the individual matrix components in biofilm resistance.

Protein Economics

Protein abundances and costs of flagellar proteins. Proteins with lower average synthetic costs (ASCs) have higher percentiles and are dark red. * = proteins with Sec secretion sequences. The number of proteins per flagellum is indicated if known.

Dan Smith was a graduate student in the lab who was interested in the evolutionary pressures on proteins.

Evolution is often thought of in functional terms. Mutations that improve or diversify a protein’s function are selected for, whereas disruptive mutations are selected against. However, economy can also play a role in protein evolution. Read all about it in Dan’s really cool paper: Economical Evolution: Microbes Reduce the Synthetic Cost of Extracellular Proteins