Background: Amyloids represent fibrillar protein aggregates with a “cross-β” spatial structure. Amyloid formation is associated with
development of more than 40 incurable diseases in humans and animals. Amyloids can also perform various physiological functions.
Most of these functional amyloids have been identified within prokaryotic species where they can act as biofilm matrix structural
components and adhesins, regulate activity of toxins, and form extracellular protein layers. While many functional amyloids are used
by pathogenic microorganisms in their interaction with multicellular hosts, little is known about the role of amyloids in host-symbiont
interactions. Previously, we have identified 54 candidate amyloid-forming proteins in the root nodule bacterium Rhizobium
leguminosarum.
Objectives: The aim of the work is to analyze candidate proteins amyloid properties in vitro and in vivo.
Methods: To analyze amyloid properties of proteins we used circular dichroism, transmission electron microscopy, fluorescence
measurement upon binding of Thioflavin T dye, polarization microscopy upon binding of Congo red dye, detergent- and proteases
resistance analysis. For analysis of localization of RopA and RopB proteins in vivo we used immunogold assay. The size and amount of
aggregates were measured with the usage of SDD-AGE.
Results: For further analysis we chose two β-barrel proteins - RopA and RopB. We demonstrated that RopA and RopB fibrils obtained in
vitro possess amyloid properties including high content of β-sheets, Thioflavin T binding, green birefringence upon staining with Congo
Red, and resistance to treatment with ionic detergents and proteases. RopA and RopB proteins aggregate in yeast cells and form Congo
Red-binding fibrils while exported to the cell surface of Escherichia coli. We demonstrated that the extracellular capsules of the R.
leguminosarum cells exhibit apple-green birefringence upon Congo Red staining. We also showed that fibrillar matrix on the cell
surface of the R. leguminosarum free-living culture binds anti-RopA and anti-RopB antibodies. Moreover, size and amount of RopA
aggregates increase after addition of flavonoid luteolin. Taking together, we may conclude that RopA and RopB proteins can form
amyloid fibrils both in vitro and in vivo. What is more, RopA is likely to be involved in early stages of symbiotic bacteria-plant
interactions as we demonstrated increase in RopA aggregation after the treatment with flavonoids.