Biofilm Formation from Secreted Proteins and Polysaccharides
Investigators passed some E. coli through a special slide chamber so they could watch at high magnification as a single bacterium attached to the surface, divided to produce a colony of a few bacteria and then began to secrete proteins (curli fibers) and polysaccharides (colanic acid and cellulose) to make the biofilm matrix. The matrix stained red with Congo Red.
Congo Red Stains Amyloids, Cellulose and Rare LPS
Staining with Congo Red shows that the spacing of hydrophobic patches on the surface of the biofilm matrix matches the flat hydrophobic, aromatic rings of the dye, Congo Red. This particular dye is important, because Congo Red also specifically stains amyloid, e.g. beta amyloid of Alzheimer’s disease. But Congo Red also binds to cellulose, a linear beta 1,4-glucan polysaccharide. This seems paradoxical, because we are taught that the sugars of which a polysaccharide are made are hydrophilic. That turns out to be a half-truth.
Faces of Sugars Are Hydrophobic
The hydrogen bonding hydroxyl groups that make sugars water soluble and hydrophilic, radiate from a ring of carbons, and the faces of that ring cannot make hydrogen bonds. The faces of sugars are hydrophobic and in most cases will bind to hydrophobic surfaces, such as aromatic amino acids, e.g. tryptophan, tyrosine and phenylalanine. Thus, carbohydrate binding enzymes, such as shown in the figure bind cellulose (in grey and red) in a groove lined with aromatic amino acids (yellow and orange) so that each sugar orients over and sometimes sandwiched between aromatic amino acid residues. This also explains why Congo Red binds to cellulose, since the aromatic rings of the dye bind to neighboring glucose residues along the relatively flat cellulose strand. Most other polysaccharides and smaller sugars lack this spacing of sugars and they don’t stain red with Congo Red.
Basic Amino Acids Bind Hydrophobically
Another misperception is that basic amino acids, positively charged lysine and arginine, are hydrophilic. The nitrogen atoms that make these amino acids positively charged, can form hydrogen bonds, but the hydrocarbon tails that have these nitrogenous tips, are hydrophobic. Thus, basic amino acids and aromatic amino acids can stack to form tryptophan/arginine ladders in which they alternate. A prominent example of these interdigitations are the way that nuclear localization signals, a quartet of basic amino acids, bind to importin via its projecting, spaced tryptophans and drag proteins through pores into the nucleus. Similarly, the basic amino acids of heparin-binding domains extend across the hydrophobic faces of the sugars of heparin and hydrogen bond with their tips to the sulfates of the heparin. In each of these binding examples the binding is primarily hydrophobic.
Amyloid Binds Congo Red by Stacked Heparin-Binding Domains
Amyloids are proteins that stack together like stacking chairs, so that each protein is oriented in the same way all along the stack. In the case of the beta amyloid that makes up the toxic plaque in Alzheimer’s disease, each amyloid peptide is stacked like a hair pin on top of the next to make a fiber. At the bend in beta amyloid, is a basic amino acid and the amyloid fiber has a band of basic amino acids along its length. The spacing between the basic amino acids in an amyloid stack is just spanned by Congo Red, so amyloids are diagnostically stained red. This same spacing of basic amino acids fits the sugars in heparin. Thus, heparin can catalyze amyloid formation and is abundant in amyloid plaques in Alzheimer’s
Bacterial Biofilms Form from Amyloids and Polysaccharides
The E. coli cells that formed the biofilms that started this article secrete a protein, curli, that stacks as an amyloid into fibers. These fibers stained by Congo Red and bind to the cellulose that is also produced by the E. coli. It should not be surprising that biofilm formation is catalyzed by heparin and biofilm formation is a major problem in catheter infection, since heparin is used to coat catheters to keep them from forming blood clots. Amyloids are also formed from stacked seminal acid phosphatase proteins that form fibers in the presence of heparin and facilitate HIV infection.
Do Biofilms Foment Amyloid Production?
Basic amino acids, sugars, aromatic amino acids and plant phytochemicals all bind each other via their hydrophobic surfaces. It would not be surprising that bacteria that produce proteins and acidic polysaccharides that interact hydrophobically would also interact with host molecules with a similar spacing of hydrophobic surfaces, which are common in heparin-binding interactions and nucleic acid interactions. The bacteria in biofilms produce both proteins and polysaccharides that may catalyze amyloid production. The acidic biofilm polysaccharide, colanic acid, may replace heparan sulfate and curli should bind to heparin.
Berberine Binds Heparin and Blocks Amyloids and Biofilms
Just as bacterial products may compete for host heparin and heparin-binding domains, phytochemicals that interact with heparin, such as the phytochemical berberine, should disrupt heparin mediated molecular interactions, and by extension also biofilms. There is experimental evidence for berberine both disrupts amyloid formation and biofilm assembly.