Background

The bacterial cell surface appendages termed curli were first discovered in an E. coli strain that was isolated from horse manure. The authors named the surface structures “curli” because of their curved appearance (Olsén et al., 1989). At nearly the same time, similar surface structures were observed in Salmonella enterica serovar Enteritidis, where they were termed thin aggregative fimbriae (Collinson et al., 1991). The curli and thin aggregative fimbriae had many unique properties, including a requirement for treatment with 90% formic acid (FA) to break the fibers apart into monomeric proteins that could be resolved by SDS-PAGE gel. A landmark study published in 1998 revealed that curli and thin aggregative fimbriae, including the genes required for biosynthesis, were virtually interchangeable between S. enterica and E. coli (Romling et al., 1998). With the publication of the genome sequence of S. Typhimurium LT2, the “curli” nomenclature was chosen for both species. In terms of function, curli are key proteins in the formation of S. enterica and E. coli biofilms, with a primary role in “short-range” cell-cell interactions leading to aggregation (Romling et al., 1998). This aggregation and biofilm formation has been linked to improved persistence and survival in the face of environmental insults (Anriany et al., 2001; Solano et al., 2002; Scher et al., 2005; Ryu and Beuchat, 2005; White et al., 2006). It is quite unique for a surface structure like curli to be conserved between these related, but long diverged species; we have proposed that curli and biofilm formation plays a key role in survival of these enteric bacteria in the environment as they cycle between their hosts. Their conservation throughout S. enterica and E. coli speaks to their importance in the lifecycle of both bacterial species.

Curli are now known to be ‘functional amyloids’. Another landmark paper showed that CsgA, the curli monomer, has self-polymerization properties that are hallmark of amyloids (Chapman et al., 2002). This explained some of the unique, biochemically resistant physical properties of curli that were observed upon their initial purification. The characteristic 3-D structure of amyloids has been termed cross-beta with regular, repeated β-sheets or strands that run perpendicular to the fiber axis. The speculation with curli is that when CsgA monomers are stacked upon each other to form fibers, there is a central non-polar core that would run along the length of curli fibers, leading to extreme stability (Collinson et al., 1999). Assembly of fibers occurs outside of the cell, where unfolded CsgA monomers pass through the outer membrane through the CsgG pore and then snap into their more rigid cross-beta structure (Evans and Chapman, 2014). Although amyloids are notoriously difficult to work with and are not easily amenable to protein crystallography because of the non-uniform fiber-fiber interactions, some structural features have been determined through the use of CsgA-specific peptides (Szulc et al., 2021). Perhaps the most striking analysis performed to date revealed that CsgA “curli” have structural similarity to the amyloid-beta fibrils that are characteristic of Alzheimer’s disease, with both sharing a characteristic structural fold called the steric β-zipper (Perov et al., 2019).

The role of curli in host-pathogen interactions has recently been clarified. Several early studies described curli in the context of host expression (Bian et al., 2000; Olsén et al., 1998; Sjobring et al., 1994), but for a long period, we thought them to be purely an “environmental” factor. Pioneering work by Çagla Tükel, Andreas Baumler, and colleagues identified purified curli fibers as potent stimulators of the innate immune system, where they interact with Toll-like receptor 2 (TLR-2) (Tükel et al., 2005), TLR1/2 and TLR1/2/9 due to the presence of extracellular DNA in complex with curli (Tükel et al., 2010; Tursi and Tükel, 2018). Systemic presentation of curli had a profound impact on autoimmunity in host species, leading to increased presence of circulating anti-dsDNA antibodies, stimulation of nod-like receptors (i.e., NLRP3), and the inflammasome (Gallo et al., 2015; Rapsinski et al., 2015). Most recently, we showed that curli are expressed by S. enterica after oral infection of mice, inside the large intestine, in both acute and long-term infection models (Miller et al., 2020). In these experiments, production of curli by S. enterica cells inside the host led to increased levels of autoimmunity and early signatures of arthritis in the knee joints of infected mice. The impact of curli was tied to the ability of S. enterica to invade host cell epithelium and cause inflammation, indicating that curli must access the lymphoid tissues surrounding the intestine to have negative effects. Many aspects of curli expression in vivo still need to be clarified.

In this manuscript, we present a series of step-by-step protocols with pictures showing how to purify these important bacterial surface structures.