Purification of the Bacterial Amyloid "Curli" from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues

Abstract

Microbiologists are learning to appreciate the importance of "functional amyloids" that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic Salmonella enterica and Escherichia coli strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from S. enterica, acknowledging that the same method can be applied to E. coli. In addition, we describe methods for detection of curli within animal tissues (i.e., GI tract) and discuss purifying curli intermediates in a S. enterica msbB mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.

Keywords: Bacterial Amyloid; Curli; Immunoblotting; Purification; Salmonella.

Video 1.. Scraping Plates

Figure 1.. Reference image of sonication apparatus used for bacterial cell lysis.

Cells are suspended in a 30 mL Oak Ridge centrifuge tube, which is embedded in ice within a 500 mL beaker. The sonicator probe is placed within the centrifuge tube approximately 5 mm beneath the surface of the liquid cell slurry.

Figure 2.. SDS-PAGE gel apparatus for curli purification from bacterial cells.

(A) Reference image of gel casting system. (B) Three milliliters of crude protein sample loaded into the well, prior to SDS-PAGE. Note: pack the SDS-PAGE gel apparatus with ice or run the experiment in a 4°C cold room to prevent gel overheating. (C) Electrophoresis is performed until the sample loading dye reaches the bottom.

Figure 3.. Collection of curli aggregates from the top of the gel after electrophoresis.

The red arrow denotes the purified curli fibrils on the top of the well before collection. Using a syringe and an 18-gauge needle, the researcher can collect the white material from the top of the gel.

Figure 4.. Lyophilization of purified curli aggregates.

(A) The solution of purified curli in water was frozen at -80°C in 30 mL Oakridge centrifuge tubes, wrapped with lint free tissue, and lyophilized for 24 h. (B) White, flocculent material (red arrow) present in the bottom of each tube after lyophilization represents curli fibrils.

Figure 5.. Quantitation and long-term storage of purified curli.

All material collected after the final lyophilization step was transferred into pre-weighed, sterile glass vials, and the rough weight was calculated. This purified material can be stored at -20°C.

Figure 6.. Comparison of different forms of curli as detected by immunoblot analysis.

(A and B) Samples were resolved by SDS-PAGE consisting of a 5% acrylamide stacking gel and 12% acrylamide resolving gel. Proteins were transferred to nitrocellulose membranes using the iBlot system. For all immunoblots, monomer (M), dimer (D), and higher molecular weight oligomers of CsgA were detected using rabbit anti-curli polyclonal serum, followed by IRDye 680RD goat anti-rabbit IgG and detection using the Odyssey CLx imaging system (Li-Cor Biosciences). (A) 20 µg and 10 µg of purified curli were depolymerized with 90% FA prior to SDS-PAGE. (B) 50 mg samples of homogenized tissues from mice infected with S. Typhimurium were treated three successive times with 90% FA prior to SDS-PAGE, as previously described ( Miller et al., 2020 ). (C) 10 µg of purified full-length curli (Mature), curli intermediates (Interm) and FA or HFIP treated curli were loaded and resolved directly on pre-cast Novex^TM 4–12% Tris-Glycine Mini Gels. The white star denotes the 25 kDa protein standard.