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.

Keywords: Entamoeba histolytica; oxidative stress resistance; queuine; queuosine; tRNA modifications.

Figure 1

Q level change determined by LC-MS/MS upon trophozoites cultivated with queuine or Q. The fold change is relative to wild type strain without any additive treatment. Data is from two biological replicates, each with three technical replicates. * indicates p value < 0.05, **** indicates p value < 0.0001, which were determined by one-way ANOVA.

Figure 2

Q level change upon trophozoites cultured with E. coli K12 vs. E. coli ΔQueC mutants. The fold change is relative to wild-type strain without any additive treatment. Data are from two biological replicates, each with three technical replicates. * indicates p value < 0.05, *** indicates p value < 0.001, **** indicates p value < 0.0001, which were determined by one-way ANOVA.

Figure 3

APB Northern blot analysis of tRNA^His_GUG in control (WT) and siEhDUF2419 trophozoites following cultivation with queuine or Q. Control (WT) or siEhDUF2419 trophozoites were cultivated in the presence of 0.1 µM queuine or Q for 3 days. (1) WT trophozoites (2) queuine-treated WT trophozoites (3) Q-treated WT trophozoites (4) siEhDUF2419 trophozoites (5) queuine-treated siEhDUF2419 trophozoites (6) Q-treated siEhDUF2419 trophozoites. Data are from two biological replicates, each with two technical replicates. * indicates p value < 0.05, ** indicates p value < 0.01. (A) APB analysis (B) quantitative analysis of relative levels of Q-tRNA^His_GUG.

Figure 4

APB Northern blot analysis of tRNA^His_GUG in control (WT) and siEhDUF2419 trophozoites that were co-cultivated with E. coli K12 or E. coli ∆QueC. Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli ∆QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) WT trophozoites (2) queuine-treated WT trophozoites (3) WT trophozoites that were cultivated with E. coli K12 (4) WT trophozoites that were cultivated with E. coli ∆QueC (5) siEhDUF2419 trophozoites (6) queuine-treated siEhDUF2419 trophozoites (7) siEhDUF2419 trophozoites that were cultivated with E. coli K12 (8) siEhDUF2419 trophozoites that were cultivated with E. coli ∆QueC (9) E. coli K12 RNA. Data are from two biological replicates, each with two technical replicates. ** indicates p value < 0.01, *** indicates p value < 0.001, **** indicates p value < 0.0001. (A) APB analysis (B) Quantitative analysis of relative levels of Q-tRNA^His_GUG.

Figure 5

Biochemical characterization of EhDUF2419. (A). In total, 10 μg of recombinant GST and GST-tagged EhDUF2419 proteins were resolved on 12% SDS gel and stain with silver staining using Pierce Silver Stain Kit according to the manufacturer’s instructions. (B). Confirmation by MS analysis of the nature of GST-EhDUF2419 protein. (C). In vitro activity of DUF2419 in Q hydrolysis into queuine. Left panel: ratio of Q UV signal and queuine MS signal; right panel. Ratio of Q MS signal (m/z 410 > 295) and queuine MS signal. Data are from two biological replicates, each with two technical replicates. ** indicated p value < 0.01.

Figure 6

EhDUD2419 expression levels in E. histolytica trophozoites. The relative fold change of EhDUD2419 expression in control (WT) and siEhDUF2419 trophozoites was calculated using the 2^{^(−ΔΔCt)} method [21]. Data are from two biological replicates, each with two technical replicates. **** indicates p value < 0.0001.

Figure 7

EhTGT level in control (WT) and siEhDUF2419 trophozoites. (A). Western blotting was performed on total protein extracts that were prepared from WT E. histolytica trophozoites (WT) and siEhDUF2419 trophozoites. The proteins were separated on 12% SDS-PAGE gels and analyzed by Western blotting using a homemade EhTGT antibody (1:1000) [12]. (B). Ponceau staining of the membrane before its incubation with EhTGT antibody. The level of EhTGT was normalized according to the total protein amount in each lane as seen by ponceau staining. Data are from two biological replicates, each with two technical replicates. ** indicates p value < 0.01.

Figure 8

Resistance to OS in queuine/Q-treated E. histolytica trophozoites. The 1 × 10⁶ control/siEhDUF2419 trophozoites that grow in the presence of 0.1 µM queuine or Q were exposed to 2.5 mM H₂O₂ for 30 min. (A). (1) Control trophozoites. (2) Control trophozoites + OS. (3) queuine-treated control trophozoites. (4) queuine-treated control trophozoites + OS. (5) Q-treated control trophozoites. (6) Q-treated control trophozoites + OS. (7) siEhDUF2419 trophozoites. (8) siEhDUF2419 trophozoites + OS. (9) queuine-treated siEhDUF2419 trophozoites. (10) queuine-treated siEhDUF2419 trophozoites + OS. (11) Q-treated siEhDUF2419 trophozoites. (12) Q-treated siEhDUF2419 trophozoites + OS. Data are from three biological replicates, each with two technical replicates. * indicates p value < 0.05. ** indicates p value < 0.01. *** indicates p value < 0.001. **** indicates p value < 0.0001. (B). (1) Control trophozoites. (2) Control trophozoites + OS. (3) Control trophozoites that were cultivated with E. coli K12. (4) Control trophozoites that were cultivated with E. coli K12 + OS. (5) Control trophozoites that were cultivated with E. coli ΔQueC. (6) Control trophozoites that were cultivated with E. coli ΔQueC + OS. (7) siEhDUF2419 trophozoites. (8) siEhDUF2419 trophozoites + OS. (9) siEhDUF2419 trophozoites that were cultivated with E. coli K12. (10) siEhDUF2419 trophozoites that were cultivated with E. coli K12 + OS. (11) siEhDUF2419 trophozoites that were cultivated with E. coli ΔQueC. (12) siEhDUF2419 trophozoites that were cultivated with E. coli ΔQueC +OS. Data are from two biological replicates, each with two technical replicates. * indicates p value < 0.05. ** indicates p value < 0.01. *** indicates p value < 0.001. **** indicates p value < 0.0001.

Figure 9

Queuine salvaging in E. histolytica. E. histolytica uptakes queuine or Q from its environment but the nature of the transporter(s) is still unknown. EhDUF2419 salvages queuine from Q or from phagocytosed E. coli cells. A study is underway to determine whether queuine salvaging occurs inside or outside of phagosomes. Next, EhTGT incorporates queuine into the wobble position of tRNA^His, tRNA^Asp, tRNA^Asn and tRNA^Tyr, which induces the expression of stress response proteins [12]. In contrast, silenced-EhDUF2419 trophozoites are not capable to salvage queuine from Q. A low level of EhDUF2419 expression impairs the level of EhTGT expression by a yet undetermined mechanism. The combination of both events (silencing of EhDUF2419 expression and low level of EhTGT expression) leads to low level of Q-tRNAs formed in the parasite and more sensitivity to OS. (Created with BioRender.com on 21 July 2022).