Queuine Salvaging in the Human Parasite Entamoeba histolytica

Abstract

Queuosine (Q) is a naturally occurring modified nucleoside that occurs in the first position of transfer RNA anticodons such as Asp, Asn, His, and Tyr. As eukaryotes lack pathways to synthesize queuine, the Q nucleobase, they must obtain it from their diet or gut microbiota. Previously, we described the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica and characterized the enzyme EhTGT responsible for queuine incorporation into tRNA. At present, it is unknown how E. histolytica salvages queuine from gut bacteria. We used liquid chromatography–mass spectrometry (LC–MS) and N-acryloyl-3-aminophenylboronic acid (APB) PAGE analysis to demonstrate that E. histolytica trophozoites can salvage queuine from Q or E. coli K12 but not from the modified E. coli QueC strain, which cannot produce queuine. We then examined the role of EhDUF2419, a protein with homology to DNA glycosylase, as a queuine salvage enzyme in E. histolytica. We found that glutathione S-transferase (GST)-EhDUF2419 catalyzed the conversion of Q into queuine. Trophozoites silenced for EhDUF2419 expression are impaired in their ability to form Q-tRNA from Q or from E. coli. We also observed that Q or E. coli K12 partially protects control trophozoites from oxidative stress (OS), but not siEhDUF2419 trophozoites. Overall, our data reveal that EhDUF2419 is central for the direct salvaging of queuine from bacteria and for the resistance of the parasite to OS.

Digestive exophagy of biofilms by intestinal amoeba and its impact on stress tolerance and cytotoxicity

“…The transfected trophozoites were then selected in TYI-S-33 medium with the addition of 3 μg/ml G418. Following selection, the trophozoites were maintained in the presence of 6 μg/ml G418, as described previously 88 . Following selection, the trophozoites were maintained in the presence of 6 μg/ml G418, as described previously 88 .…”

Structural basis of Qng1-mediated salvage of the micronutrient queuine from queuosine-5′-monophosphate as the biological substrate

“…Recently, two groups reported a weak in vitro activity of DUF2419 from S. pombe ( 37 ) and from the human parasite Entamoeba histolytica ( 38 ) , that converts queuosine to queuine, and one assigned it the function of ‘queuosine nucleoside glycosylase.’ Here, we report comprehensive functional, structural and biochemical characterization of S. thermophilus and human DUF2419, and show with in vitro and in vivo evidence that DUF2419 is a q ueuosine- n ucleotide N- g lycosylase/hydrolase (Qng1 for the bacterial enzyme, and QNG1 for the mammalian ortholog C9orf64), that releases q from Q-5′-monophosphate as the biological substrate, and this activity is required for the salvage of q from exogenous Q by S. pombe and human cells. Notably, in human cells Q-5′-monophosphate accumulates intracellularly in the absence of QNG1, indicating intracellular conversion of imported Q to the 5′-nucleotide by an unidentified kinase.…”