Fluorescent Labeling of Helminth Extracellular Vesicles Using an In Vivo Whole Organism Approach

Abstract

In the last two decades, extracellular vesicles (EVs) from the three domains of life, Archaea, Bacteria and Eukaryotes, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in Anisakis spp. and Trichuris suis larvae. EVs isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study, therefore, presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species.

Keywords: Cryo–EM; extracellular vesicles; helminth; proteomics; vesicle labelling; vesicle tracking.

Figure 1

Hatched T. suis (L1) cultured for 4 h in the presence of 4 µM DOPE-Rho (A,B), control media (C,D) and dead T. suis (L1) cultured in 4 µM DOPE-Rho (E,F) prior to washing, fixation with 4% paraformaldehyde and analysis using fluorescence microscopy. Orange = Rhodamine, Blue = Hoechst nuclear stain, Scale bar: 100 µm.

Figure 2

Anisakis spp. L3 cultured for 16 h in the presence of 0 µM (A), 1 µM (B) or 4 µM (C–F) DOPE-Rho prior to washing, fixation with 4% paraformaldehyde and analysis using fluorescence microscopy. Orange = Rhodamine, Scale bar: 100 µm. Corresponding bright-field images are inset.

Figure 3

Nanoparticle tracking analysis (NTA) of 100,000× g pellets from untreated Anisakis spp. (A), 1 µM (B), 4 µM (C) and 8 µM DOPE-Rho incubated Anisakis spp. (D). NTA analysis of no worm dye control (NWC) 1 µM (E) and 4 µM (F) 100,000× g pellets. Particle number per ml for controls and DOPE-Rho incubated Anisakis spp. (G). Relative fluorescent intensity (R:F:I) of 100,000× g pellets assessed at excitation 525 nm and emission 565–615 nm (H).

Figure 4

Cryo-TEM images of 1 µM DOPE-Rho labelled Anisakis spp. vesicles. Scale bar: 100 nm.

Figure 5

PMA differentiated THP-1 cells incubated overnight with 2 µg (A–C), or 8µg (D,E and Figure 1). µM DOPE-Rho labelled EVs. THP-1 cells incubated with 12 µg non-labelled EVs (G,H) or PBS control (I). Orange = Rhodamine labelled EVs, Blue = Hoechst nuclear stain. Scale bar: 100 µm.