PH-triggered endosomal escape of pore-forming Listeriolysin O toxin-coated gold nanoparticles

Ismael Plaza-Ga,Víctor Almendro-Vedia,Vanesa Manzaneda-González,Matic Kisovec,Iván López Montero,Andrés Guerrero-Martínez,Paolo Natale,Gregor Anderluh,Mónica Muñoz-Úbeda

doi:10.1186/s12951-019-0543-6

Abstract

BackgroundA major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell.ResultsAs a “proof of concept”, we present here the use of purified His-LLO H311A mutant protein and its conjugation on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fibroblasts. Surface immobilization of LLO was achieved after specific functionalization of the nanoparticles with nitrile acetic acid, enabling the specific binding of histidine-tagged proteins.ConclusionsEndosomal acidification leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.

Highlights

A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest
We observed stimulated pore-formation activity of Listeriolysin O (LLO) H311A at pH 5 and moderate activity at pH 6–8 (Fig. 1c), while no strong pH-dependent pore formation was observed in the case of the LLO wild type protein (Additional file 1: Figure S1)
In the presence of LLO, we observed the attachment of sedimented PC/ Chol-giant unilamellar vesicles (GUVs) to the supported lipid bilayer (SLB) layer, as indicated by the reduced GUV mobility on the SLB surface. This local attachment occurs most likely through binding of PC/Chol-GUV to the cholesterol binding motif of the surface LLO protein (Additional file 1: Figure S2A), which was not observed in the absence of LLO (Additional file 1: Figure S2B), where the position of the sedimented PC/Chol-GUVs was found to change during confocal laser scanning microscopy (CLSM) imaging

Summary

Results

As a “proof of concept”, we present here the use of purified His-LLO H311A mutant protein and its conjuga‐ tion on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fibroblasts. Surface immobilization of LLO was achieved after specific functionalization of the nanoparti‐ cles with nitrile acetic acid, enabling the specific binding of histidine-tagged proteins

Conclusions

Background

Results and discussion

Conclusion

Methods