TY - CHAP

T1 - In Vitro And In Vivo Amyloid Formation By The RopA And RopB Proteins From Rhizobium Leguminosarum

AU - Kosolapova, A. O.

AU - Belousov, M. V.

AU - Sulatskaya, A. I.

AU - Belousova, M. E.

AU - Sulatsky, M. I.

AU - Antonets, K. S.

AU - Volkov, K. V.

AU - Lykholay, A. N.

AU - Shtark, O. Y.

AU - Vasileva, E. N.

AU - Zhukov, V. A.

AU - Ivanova, A. N.

AU - Zykin, P. A.

AU - Kuznetsova, I. M.

AU - Turoverov, K. K.

AU - Tikhonovich, I. A.

AU - Nizhnikov, A. A.

PY - 2021/6/24

Y1 - 2021/6/24

N2 - Background: Amyloids represent fibrillar protein aggregates with a “cross-β” spatial structure. Amyloid formation is associated withdevelopment 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 structuralcomponents and adhesins, regulate activity of toxins, and form extracellular protein layers. While many functional amyloids are usedby pathogenic microorganisms in their interaction with multicellular hosts, little is known about the role of amyloids in host-symbiontinteractions. Previously, we have identified 54 candidate amyloid-forming proteins in the root nodule bacterium Rhizobiumleguminosarum.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, fluorescencemeasurement upon binding of Thioflavin T dye, polarization microscopy upon binding of Congo red dye, detergent- and proteasesresistance analysis. For analysis of localization of RopA and RopB proteins in vivo we used immunogold assay. The size and amount ofaggregates 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 invitro possess amyloid properties including high content of β-sheets, Thioflavin T binding, green birefringence upon staining with CongoRed, 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 cellsurface of the R. leguminosarum free-living culture binds anti-RopA and anti-RopB antibodies. Moreover, size and amount of RopAaggregates increase after addition of flavonoid luteolin. Taking together, we may conclude that RopA and RopB proteins can formamyloid fibrils both in vitro and in vivo. What is more, RopA is likely to be involved in early stages of symbiotic bacteria-plantinteractions as we demonstrated increase in RopA aggregation after the treatment with flavonoids.

AB - Background: Amyloids represent fibrillar protein aggregates with a “cross-β” spatial structure. Amyloid formation is associated withdevelopment 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 structuralcomponents and adhesins, regulate activity of toxins, and form extracellular protein layers. While many functional amyloids are usedby pathogenic microorganisms in their interaction with multicellular hosts, little is known about the role of amyloids in host-symbiontinteractions. Previously, we have identified 54 candidate amyloid-forming proteins in the root nodule bacterium Rhizobiumleguminosarum.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, fluorescencemeasurement upon binding of Thioflavin T dye, polarization microscopy upon binding of Congo red dye, detergent- and proteasesresistance analysis. For analysis of localization of RopA and RopB proteins in vivo we used immunogold assay. The size and amount ofaggregates 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 invitro possess amyloid properties including high content of β-sheets, Thioflavin T binding, green birefringence upon staining with CongoRed, 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 cellsurface of the R. leguminosarum free-living culture binds anti-RopA and anti-RopB antibodies. Moreover, size and amount of RopAaggregates increase after addition of flavonoid luteolin. Taking together, we may conclude that RopA and RopB proteins can formamyloid fibrils both in vitro and in vivo. What is more, RopA is likely to be involved in early stages of symbiotic bacteria-plantinteractions as we demonstrated increase in RopA aggregation after the treatment with flavonoids.

UR - https://fems-microbiology.org/wp-content/uploads/2021/10/FEMS-Abstract-Book-WMF.pdf

M3 - Conference abstracts

BT - World Microbe Forum: FEMS

Y2 - 20 June 2021 through 24 June 2021

ER -