Two Novel Amyloid Proteins, RopA and RopB, from the Root Nodule Bacterium Rhizobium leguminosarum

Abstract

Amyloids represent protein fibrils with a highly ordered spatial structure, which not only cause dozens of incurable human and animal diseases but also play vital biological roles in Archaea, Bacteria, and Eukarya. Despite the fact that association of bacterial amyloids with microbial pathogenesis and infectious diseases is well known, there is a lack of information concerning the amyloids of symbiotic bacteria. In this study, using the previously developed proteomic method for screening and identification of amyloids (PSIA), we identified amyloidogenic proteins in the proteome of the root nodule bacterium Rhizobium leguminosarum. Among 54 proteins identified, we selected two proteins, RopA and RopB, which are predicted to have β-barrel structure and are likely to be involved in the control of plant-microbial symbiosis. We demonstrated that the full-length RopA and RopB form bona fide amyloid fibrils in vitro. In particular, these fibrils are β-sheet-rich, bind Thioflavin T (ThT), exhibit green birefringence upon staining with Congo Red (CR), and resist treatment with ionic detergents and proteases. The heterologously expressed RopA and RopB intracellularly aggregate in yeast and assemble into amyloid fibrils at the surface of Escherichia coli. The capsules of the R. leguminosarum cells bind CR, exhibit green birefringence, and contain fibrils of RopA and RopB in vivo.

Accumulation of storage proteins in plant seeds is mediated by amyloid formation

“…The best results have been obtained by the usage of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) solvent at 37˚C for the proteins dissolution with its subsequent removal from the sample and incubation of dissolved proteins in the distilled water ( Fig 1B, row 4). Similar conditions have been used previously in other works to obtain amyloid fibrils of human amyloid-beta peptide (Aβ) [33] and RopA and RopB proteins of the bacterium Rhizobium leguminosarum [34].…”

“…Notably, being structurally similar to Cupins, β-barrel domain-containing proteins that belong to the outer membrane proteins (Omps) of Gram-negative bacteria also form amyloids [34,70,71] whereas human Aβ (1-42 aa) vice versa forms β-barrel pores in specific conditions [72]. In addition, recently, formation of β-barrel oligomer intermediates has been proposed as a common step in general mechanism of amyloid formation [73].…”

Biological Functions of Prokaryotic Amyloids in Interspecies Interactions: Facts and Assumptions

“…In vitro, RopA and RopB form fibrillar aggregates exhibiting typical physicochemical properties of amyloids including green birefringence in polarized light upon CR staining, binding of ThT, resistance to proteases and ionic detergents treatment. In vivo, RopA and RopB form extracellular amyloid fibrils after the prolonged incubation of R. leguminosarum cells on the cultural media [ 40 ]. Moreover, the amount and size of the aggregates formed by RopA protein after induction of the nodulation process in free-living culture is increased after flavonoid treatment [ 40 ].…”

“…In vivo, RopA and RopB form extracellular amyloid fibrils after the prolonged incubation of R. leguminosarum cells on the cultural media [ 40 ]. Moreover, the amount and size of the aggregates formed by RopA protein after induction of the nodulation process in free-living culture is increased after flavonoid treatment [ 40 ]. Based on these observations, RopA and RopB hypothetically act as adhesins and represent a part of the EPS of biofilms formed by R. leguminosarum on different surfaces including the plant roots, thus participating in colonization of plant by bacteria cells.…”

“…Some recent demonstration of amyloid properties of RopA and RopB proteins of symbiotic root nodule bacterium Rhizobium leguminosarum bv. viciae [ 40 ] suggests that the bacterial amyloids can be involved not only in the interactions between pathogenic bacteria and multicellular hosts but also in the symbiotic (Type III) interactions with them. Notably, the virulence mechanisms—the ability of a microorganism to successfully infect and colonize host—share similarities between symbiotic bacteria belonging to the order Rhizobiales and pathogenic microorganisms [ 177 ].…”