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

Kosolapova Kosolapova,Sulatsky Sulatsky,Zykin Zykin,Antonets Antonets,Vasileva Vasileva,Sulatskaya Sulatskaya,Belousova Belousova,Belousov Belousov,Zhukov Zhukov,Turoverov Turoverov,Shtark Shtark,Nizhnikov Nizhnikov,Ivanova Ivanova,Lykholay Lykholay,Volkov Volkov,Tikhonovich Tikhonovich,Kuznetsova Kuznetsova

doi:10.3390/biom9110694

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.

Highlights

Amyloids are fibrillar protein aggregates characterized by a highly ordered spatial structure called cross-β [1,2,3]
Based on data of UniProt, the majority of these proteins are cytoplasmically located and functionally related to metabolic enzymes, while membrane proteins comprise about one-fifth of their number (Figure 1A) The identified proteins were predicted to act as porins and transporters, ribosomal proteins, DNA repair enzymes, and chaperonins (Figure 1A)
On the basis of analysis of the Thioflavin T (ThT) interaction with amyloid fibrils formed from a wide range of proteins and peptides, we proposed in our works that the second type of dye binding occurs during the clumping of amyloids

Summary

Introduction

Amyloids are fibrillar protein aggregates characterized by a highly ordered spatial structure called cross-β [1,2,3]. Amyloid fibrils are formed by intermolecular β-sheets stabilized by numerous hydrogen bonds that give rise to a cross-β X-ray diffraction pattern [2,3]. This type of structure determines amyloid stability, resulting in their resistance to treatment with protein denaturants like ionic detergents [4], as well as proteases [5]. About 40 amyloid-forming proteins and peptides associated with the development of incurable human and animal diseases were discovered [1]. In fungi, amyloids are involved in the control of heterokaryon incompatibility [8], cell adhesion [9], spore formation and dispersal [10], and multicellularity [11]

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Conclusion