Entamoeba-derived Exosome Research and Application

1. Culture Entamoeba histolytica trophozoites axenically under standard conditions, then inoculate the chilled trophozoites at the mid-log phage in encystation media to obtain encysting Entamoeba histolytica.
2. Amplify Entamoeba histolytica until confluence and then incubate overnight under anaerobic conditions.
3. Collect Entamoeba histolytica media carefully, and initially purify the media by centrifugation at low speed and filtration.
4. Overnight sediment exosomes in Entamoeba histolytica media with chemical reagents, followed by centrifugation to obtain Entamoeba-derived exosome precipitates.

• Validation of Entamoeba-derived exosomes carrying protein cargo.

Comparison of proteins in Entamoeba-derived exosomes and whole-cell lysates by protein gel electrophoresis showed that Entamoeba-derived exosomes were selectively enriched and depleted of some proteins. Gal/GalNAc lectin proteins were selectively enriched in exosomes, while cytosolic skeleton proteins were selectively depleted.

• Proteomic profiling of Entamoeba-derived Exosome

Mass spectrometry profiling and GO analysis showed that Entamoeba-derived Exosome proteins hardly contain proteins associated with the plasma membrane, or compartments other than endosomes, but mainly encompass proteins associated with vesicle genesis, molecular binding and signaling.

• RNA profiling of Entamoeba-derived Exosome.

PAGE assay revealed that Entamoeba-derived exosomes were selectively enriched for some 27-nt small RNAs and were able to protect the RNA cargo from RNase A digestion. Moreover, the presence of argonaute 2-2 protein and Tudor protein, which bind to small RNAs to mediate target gene silencing, was also confirmed in Entamoeba-derived exosomes by protein blotting, suggesting that these exosomal small RNAs mediate the RNAi process.

• Functional assay of Entamoeba-derived exosomes.

The extent of the encystation process was indicated by luciferase signaling. Encysting Entamoeba-derived exosomes promoted E. invadens encystation, whereas exosomes derived from metabolically active Entamoeba trophozoites impeded encystation, suggesting a function for Entamoeba-derived exosomes to mediate parasite-to-parasite communication.

• Entamoeba-derived exosomes affected ROS levels in neutrophils.

The ROS levels released in neutrophils were detected by a ROS fluorescent probe, and it was found that compared to neutrophil-derived exosomes and Entamoeba-derived exosomes, exosomes derived from co-cultures of neutrophils and Entamoeba histolytica trophozoites were capable of transfer higher levels of ROS.

• Entamoeba-derived exosomes suppressed the respiratory burst and NET of neutrophils.

Pretreatment with Entamoeba-derived exosomes was found to significantly reduce chemical and biological stimulant-induced oxidative burst and NETosis in neutrophils by fluorescence quantification, whereas co-culture-derived exosomes were able to more substantially delay NETosis and reduce NET release.