Research in the Chapman Lab

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.

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. Our work blends 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.

Curlin Assembly

Curlin assembly in E. coli requires the products of at least five genes located on two divergently transcribed operons (Fig. 2). On the cell surface, or possibly during secretion through the outer membrane (OM), the major curlin subunit, CsgA, is nucleated into fibers by the minor curlin subunit, CsgB. This nucleation reaction is the crux of curli (and eukaryotic amyloid) formation, and understanding the molecular details of nucleation is a focus of our laboratory. In the absence of the CsgB nucleator, CsgA is secreted from the cell in a soluble, unassembled state. However, soluble CsgA can polymerize into curli fibers if it contacts an adjacent cell expressing the CsgB nucleator, in a process called interbacterial complementation (Fig. 3).

One model of curli polymerization suggests that CsgB induces a conformational change in CsgA that nucleates its assembly into fibers. Polymerized CsgA would then induce a similar change in the next incoming CsgA subunit, and repetition of this process would drive curli assembly. Such a model assumes that conformationally altered CsgA would have a nucleator activity similar to that of CsgB. Interestingly, CsgA and CsgB are proteins of identical predicted size, are built up of similar repeat motifs, and exhibit 49% sequence similarity. We have purified CsgA in a soluble, unassembled state from cells lacking CsgB. Soluble CsgA does not adopt the ? sheet-rich structure or the fibrous appearance that typify curli. After prolonged incubation CsgA spontaneously assembles into fibers that bind Congo red, demonstrating that CsgB is not absolutely required for nucleation and polymerization of CsgA. At lower CsgA concentrations (and, in vivo) CsgA self-polymerization is inefficient. We are using this in vitro system to further investigate the role of CsgB-mediated nucleation.

A comprehensive bank of CsgA mutants is being constructed and cloned into expression vectors for purification to help define the sequences necessary for polymerization and nucleation of CsgA. This work has far-reaching implications. Previously, amyloid fiber formation was thought to be a biological blunder, and virtually nothing is known about how eukaryotic amyloid formation is nucleated in vivo. By understanding and exploiting the elegant biogenesis pathway that E. coli uses to produce curli fibers, strategies to prevent or reverse eukaryotic amyloid formation can be developed.

Protein Export

We are investigating how curlin proteins are exported. Both CsgA and CsgB are transported to the cell surface under the guidance of three curlin accessory proteins, CsgE, CsgF, and CsgG. CsgG is a lipoprotein, localized to the periplasmic side of the outer membrane (Fig. 2). Our work has shown that CsgG oligomerizes in the OM into a barrel-like structure with an apparent central pore of 2 nm. This structure is the backbone of the curli subunit secretion apparatus, and without it CsgA and CsgB are confined to the periplasmic space where they are proteolytically degraded. Secretion of CsgA is guided by the 22 N-terminal amino acids on mature CsgA that form a specific CsgG-dependent secretion signal.

We have developed and optimized several assays that will aid in furthering our knowledge about curlin subunit secretion across the OM. First, CsgG-mediated pore formation can be measured using a simple antibiotic sensitivity assay, and a bank of CsgG mutants has been screened for pore-forming ability using this assay. CsgG can be routinely purified from the OM of bacteria, and structural studies on this unique protein will continue in the laboratory. CsgG forms an assembly platform in the OM and other curli proteins, including CsgE and CsgF, can be localized to OM in a CsgG-dependent fashion. CsgE and CsgF are type III chaperone-like proteins that contribute to the stability and proper localization of the CsgA and CsgB subunit proteins. Co-purification experiments demonstrated that CsgE and CsgG physically interact, consistent with the idea that CsgE chaperones CsgG to the OM. Delineating the structural basis and result of the CsgE-CsgG interaction will continue to be a focus of the laboratory.

Host-Bacteria Interactions

We are also interested in understanding the biological benefit of bacterial amyloid production. Bacterial species that express curli (E. coli and Salmonella spp.) are the determinants of many human diseases. Curli are peritrichously arranged on the cell surface, leaving them perfectly situated to mediate interactions at the host-bacteria interface. Consistent with this, curli bind to many host proteins and could potentially act as bacterial adhesion molecules during the initial steps of infection.

Host epithelial cells, as well as dedicated immune surveillance cells such as macrophages, efficiently recognize curliated bacteria and elicit a potent immune response to clear the bacteria. However, the nature of this response is not known. Curli may augment the response to another bacterial factor, such as LPS, or they might stimulate the immune system directly.