Stress Response in Entamoeba histolytica Is Associated with Robust Processing of tRNA to tRNA Halves

tRNA-derived fragments have been reported in many different organisms and have diverse cellular roles, such as regulating gene expression, inhibiting protein translation, silencing transposable elements, and modulating cell proliferation. In particular, tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here, we report the presence of tRNA-derived fragments in Entamoeba, with tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite-to-cyst conversion, with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves, as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments associated with Entamoeba Argonaute proteins, EhAgo2-2 and EhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoebas. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses, including encystation, suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments in Entamoeba. IMPORTANCE In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small-RNA sequencing data sets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during the developmental process of encystation. We also found that shorter tRNA-derived fragments are bound to Entamoeba Argonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway, which mediates robust gene silencing in Entamoeba. We noticed that in response to heat shock, the protein translation levels were elevated in the parasites. This effect was reversed in the presence of an analog of leucine, which also reduced the levels of the tRNA halves in the stressed cells. Our results suggest that tRNA-derived fragments in Entamoeba have a possible role in regulating gene expression during environmental stress.