Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Oct 25;28(21):7262.
doi: 10.3390/molecules28217262.

The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation

Paulina Aguilera  1 , Camilo Berríos-Pastén  1 , Marcelo Veloso  1 , Matías Gálvez-Silva  1 , Florian Turbant  2   3 , Rosalba Lagos  1 , Frank Wien  2 , Veronique Arluison  3   4 , Andrés E Marcoleta  1
Affiliations

The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation

Paulina Aguilera et al. Molecules. .

Abstract

Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the β-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

Keywords: EGCG; bacterial amyloid; green tea; microcin E492; synchrotron-radiation circular dichroism.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of EGCG on MccE492 amyloid formation monitored by ThT fluorescence. (A) Schematic representation of the typical amyloid aggregation kinetics followed by ThT. (B) MccE492 (200 µg/mL) aggregation kinetics in PIPES aggregation buffer, with or without 1 mM EGCG. (C) MccE492 (400 µg/mL) aggregation kinetics in phosphate buffer, with or without 1 mM EGCG. Amyloid formation was monitored by measuring ThT fluorescence (excitation: 450 nm; emission: 482 nm) every 15 s for 70 h at 37 °C. A.U. = arbitrary units. The curves are representative of three independent experiments with each buffer.
Figure 2
Figure 2
EGCG promotes a soluble state of MccE492 and impairs the formation of higher-order aggregates. Purified MccE492 (200 µg/mL) was incubated in the absence or presence of 1 mM EGCG in PIPES aggregation buffer at 37 °C. The soluble protein was visualized by immunoblot after centrifuging the samples at 16,000× g for 30 min and recovering the supernatant. A monoclonal anti-MccE492 antibody was used. MccE492 monomers (~10 kDa) and oligomers (>10 kDa) are pointed out. MW: molecular weight standard (kDa). Although MccE492 has a molecular weight close to 8 kDa, it has anomalous electrophoretic migration, with an apparent MW of 10 kDa.
Figure 3
Figure 3
Visualization of MccE492 amyloid fibers morphology in the absence and presence of EGCG. Purified MccE492 (200 µg/mL) was incubated with or without 1 mM EGCG in PIPES aggregation buffer at 37 °C. At the indicated times, samples were collected and visualized by negative-stain electron microscopy. Scale bar: 200 nm. Images are representative of three independent experiments. Additional images are shown in Figure S1.
Figure 4
Figure 4
Effect of EGCG on MccE492 amyloid secondary structure signature and thermal stability, as revealed by SRCD. SRCD spectra of MccE492 (2 mg/mL) in phosphate buffer incubated at 37 °C in the absence (A) or presence (B) of 1 mM EGCG. (C,D) SRCD thermal scan of preformed amyloid fibers without (C) or with (D) EGCG 1 mM.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Eisenberg D.S., Sawaya M.R. Structural Studies of Amyloid Proteins at the Molecular Level. Annu. Rev. Biochem. 2017;86:69–95. doi: 10.1146/annurev-biochem-061516-045104. - DOI - PubMed
    1. Chiti F., Dobson C.M. Protein Misfolding, Amyloid Formation, and Human Disease: A Summary of Progress Over the Last Decade. Annu. Rev. Biochem. 2017;86:27–68. doi: 10.1146/annurev-biochem-061516-045115. - DOI - PubMed
    1. Fowler D.M., Koulov A.V., Balch W.E., Kelly J.W. Functional Amyloid—From Bacteria to Humans. Trends Biochem. Sci. 2007;32:217–224. doi: 10.1016/j.tibs.2007.03.003. - DOI - PubMed
    1. Van Gerven N., Van der Verren S.E., Reiter D.M., Remaut H. The Role of Functional Amyloids in Bacterial Virulence. J. Mol. Biol. 2018;430:3657–3684. doi: 10.1016/j.jmb.2018.07.010. - DOI - PMC - PubMed
    1. Levkovich S.A., Gazit E., Laor Bar-Yosef D. Two Decades of Studying Functional Amyloids in Microorganisms. Trends Microbiol. 2021;29:251–265. doi: 10.1016/j.tim.2020.09.005. - DOI - PubMed

LinkOut - more resources

-