EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2

Abstract

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils in solution. As this propensity of MSP2 to form fibrils in solution has the potential to impede its development as a vaccine candidate, finding an inhibitor that inhibits fibrillogenesis may enhance vaccine development. We have shown previously that EGCG inhibits the formation of MSP2 fibrils. Here we show that EGCG can alter the β-sheet-like structure of the fibril and disaggregate pre-formed fibrils of MSP2 into soluble oligomers. The fibril remodelling effects of EGCG and other flavonoids were characterised using Thioflavin T fluorescence assays, electron microscopy and other biophysical methods.

Fig. 1

Effect of flavonoids on ThT fluorescence of MSP2 fibrils. Kinetics was measured in the presence of EGCG, baicalein and resveratrol. FC27 MSP2-6H (40 µM) was incubated in the presence of a 10-fold molar excess of EGCG and a 15-fold molar excess each of baicalein and resveratrol, and ThT fluorescence was measured over a period of 22 h.

Fig. 2

Analysis of MSP2 fibril morphology by TEM after negative staining with 2% uranyl acetate. FC27 NT-MSP2 fibrils after incubation for 24 h in A) PBS, B) 1.5% DMSO, C & D) a 10-fold molar excess of EGCG, and a 15-fold molar excess of E) baicalein and F) resveratrol. D) Globular aggregates of MSP2 are components of the larger amorphous aggregates.

Fig. 3

EGCG binds directly to MSP2 fibrils. Comparison of A) Nitroblue tetrazolium (NBT)-stained nitrocellulose membrane and B) Coomassie-stained SDS-PAGE gel of MSP2 samples. FC27 NT-MSP2 was incubated for one week in the presence and absence of EGCG (molar ratios of EGCG to MSP2 are indicated above the gel).

Fig. 4

EGCG alters the structure of MSP2 fibrils. Far-UV CD spectra of FC27 MSP2-6H were measured in the absence and presence of A) equimolar and B) 10-fold excesses of EGCG. CD spectra were recorded for the samples after incubation with EGCG for 0, 1 and 24 h.