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Parasites release extracellular vesicles (EVs) which, in some cases, modulate the host’s immune response contributing to the establishment of the infection. In this work we have isolated and characterized the EVs released by trophozoites of the human protozoan parasite Entamoeba histolytica, the causal agent of amoebiasis, when alone or in coculture with human neutrophils, and determined their effect on neutrophil NETs and ROS production. Nanoparticle tracking analysis showed that amoebic EVs are variable in size, ranging from less than 50 nm to nearly 600 nm in diameter (average of 167 nm), whereas neutrophil EVs are more uniform in size, with an average of 136 nm. In cocultures amoeba:neutrophil (1:100) most EVs are 98 nm in size, which is the typical size of exosomes. EVs from amoebae and neutrophils showed almost equal levels of ROS, which were considerably increased in EVs from cocultures. Uptake of amoebic EVs by neutrophils was demonstrated by fluorescence and resulted in a significant reduction in the oxidative burst and NET release triggered by PMA, ionophore A23187, or the amoebae itself used as stimuli. Interestingly, uptake of EVs from cocultures did not affect ROS production, but instead caused a greater delay in the onset of NETs release and in their quantity. A comparative proteomic analysis between the EVs of amoebae and neutrophils separately vs the cocultures showed a similar distribution of protein categories in the GO analysis, but differences in the expression and abundance of proteins such as the N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin and calreticulin in amoeba EVs, and various antimicrobial molecules in neutrophil EVs, such as lactoferrin and myeloperoxidase. These results highlight the importance of EVs in the immunomodulatory effects exerted by amoeba on human neutrophils.

Predicted secondary structure of the tRNA-Ala (panel a) and the tRNA-like associated to the L. major U2 snRNA gene (panel b). The minimum free energy structures are shown. The color scale indicates low (blue) to high (red) probabilities of base pairing. (PDF 355 kb)

Mutations introduced in the tRNA-Ala/U2 snRNA locus. For each vector, sequence elements are indicated inside colored boxes: tRNA-Ala in green, tRNA-like in orange and U2 snRNA in blue. Boxes A and B are also indicated. The tag sequence, inserted within the U2 snRNA gene, is also shown. Base substitutions are indicated in red font. (PDF 3122 kb)

Sequence comparisons of U2 snRNA genes and flanking regions from T. cruzi (CL Brener Non-Esmeraldo-like). Sequences from the genes located on chromosomes 23, 37 and 6 are shown. The U2 snRNA gene from chromosome 23 is presented in blue font. The position of boxes A and B is indicated. Sequence numbers are relative to the TSS (+1) from the U2 snRNA. (PDF 1404 kb)

Sequence alignment of U2 snRNA genes from trypanosomatids. Sequences were obtained from the TriTrypDB databases of the following species of trypanosomatids: L. major Friedlin (Lmj), L. arabica LEM1108 (Lar), L. tarentolae Parrot-TarII (Ltr), L. infantum JPCM5 (Lin), L. amazonensis MHOMBR/71973/M2269 (Laz), L. donovani BPK282A1 (Ldn), L. aethiopica L147 (Lae), L. mexicana U1103 (Lmx), L. panamensis MHOM/COL/81/L13 (Lpn), L. braziliensis M2904 (Lbr), L. enriettii LEM3045 (Len), E. monterogeii LV88 (Emg), C. fasciculata Cf-Cl (Cfs), L. pyrrhocoris H10 (Lpy), L. seymouri ATCC 30220 (Lsy), L. collosoma (unspecified strain) [39] (Lcl), T. cruzi CL Brener Non-Esmeraldo-like, copy of the chromosome 23 (Tcr), T. vivax Y486 (Tvx), T. congolense IL3000 (Tcn), T. evansi STIB 805 (Tev) and T. brucei DAL972 (Tbr). The alignment was obtained with the DNAMAN software, and manually corrected. Arrows indicate the identified pseudouridines (Ψ) in the L. major U2 snRNA (this work). BPRR and Sm sites are also indicated. The 3' ends (C residues) that have been experimentally mapped are shown in red font for L. major (this work), T. cruzi [45] and T. brucei [44]. The T-tract length is indicated for each species. Sequence numbers are relative to the TSS (+1) from the U2 snRNA. This alignment was used to generate the sequence logo depicted in Fig. 3b. (PDF 2137 kb)