Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites

Abstract

The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNA(Gln) and a glutaminyl-tRNA amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln). Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyl-tRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNA(Gln) to Gln-tRNA(Gln) in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria.

Keywords: apicoplast; glutamyl-tRNA amidotransferase; glutamyl-tRNA synthetase; malaria; nucleic acid; nucleic acid enzymology; plasmodium; transfer RNA (tRNA).

FIGURE 1.

PfGatAB tertiary structure reveals conserved functional features. To predict the tertiary structure and identify conserved and divergent features of PfGatAB, homology modeling was performed using the S. aureus GatCAB crystal structure as a template (28, 29). The PfGatA (orange) and PfGatB (green) core is highly conserved, and most functional residues are present. Glutamine (cyan sticks), ADP (yellow sticks), and Mg²⁺/Mn²⁺ ions (magenta/purple ball) are taken from superimposed GatCAB structures (PDB codes 2f2a, 2g5i, and 2df4). Residues involved in binding are drawn as sticks, and their labels are colored analogously to the binding partner. The active site of PfGatA comprises the glutamine-binding site (cyan), the conserved Ser-cis-Ser-Lys catalytic scissors, shown in a stick representation and marked with yellow labels, and the conserved oxyanion hole (red labels) that stabilizes the tetrahedral covalent intermediate. GatB contains a cradle domain (dark green), which binds the substrate, ADP, and Mg²⁺/Mn²⁺ and a helical domain (light green). The region (amino acids 805–880) in PfGatB is moveable and is probably involved in tRNA recognition. The active sites of PfGatA and PfGatB are presumably connected by a conserved ammonia channel, with Thr³⁴³ (red/blue label) at the entrance and Lys⁴¹⁰ (blue label) at the exit of the tunnel. We could not find any homologous structure for the two inserts in PfGatA (425–530 and 708–744, pale orange) or for the inserts in PfGatB (461–525, pale green). Furthermore, no template was found for the first 180 N-terminal residues in PfGatA and the first 349 N-terminal residues in PfGatB.

FIGURE 2.

Phylogenetic analysis of the Plasmodium apicoplast GatB and GatA proteins. A, maximum likelihood phylogenetic relationship between all Plasmodium GatB sequences and GatB and GatE sequences from bacteria and archaea, respectively. If Plasmodium GatB evolved from GatB or GatE, one would expect the phylogenetic pattern to show these enzymes to be specifically related to a particular GatB or GatE clade; instead, the resulting phylogenetic trees had three branches representing three distinct subfamilies with Plasmodium GatB being highly distinct and separated from the other plastid GatB, bacterial GatB and archaeal GatE clades. Scale bar, 0.5 changes/site B, maximum likelihood phylogenetic tree of Plasmodium GatA sequences and GatA and GatD sequences from bacteria and archaea. The resulting phylogenetic tree also had three branches representing three distinct subfamilies with Plasmodium GatA placed on a separate branch away from bacterial GatA, archaeal GatD, or the plastid GatA clades. Scale bar, 0.9 changes/site.

FIGURE 3.

PfGatA and PfGatB localizes to the apicoplast. Transgenic PfGatA-GFP and PfGatB-GFP parasites were generated in which an episomal construct contained the bipartite apicoplast targeting sequences from PfGatA and PfGatB were cloned in front of a GFP, and the expression was controlled by the P. falciparum calmodulin 5′ promoter (35). The transfection experiments were performed in duplicate and repeated once. Differential interference contrast and fluorescent images were captured and processed using deconvolution microscopy; a merge of the images is presented on the far right column (overlay). A and B, PfGatA-GFP and PfGatB-GFP apicoplast localization was monitored via immunofluorescence assay using an anti-GFP antibody (green), and the apicoplast was detected by staining it with anti-ACP antibody (red). Nucleic acid was stained with DAPI (blue). PfGatA-GFP and PfGatB-GFP form characteristically small and round compartments early in the infection cycle (A, panel i, and B, panel i), which then elongate and develop into complex and multiply branched forms at the trophozoite stage prior to splitting into individual spots, one for each merozoite, in the schizont stage (A, panel ii, and B, panel ii). PfGatA-GFP and PfGatB-GFP co-localized with ACP (α-GFP/α-ACP overlay), confirming localization to the plastid. C and D, PfGatA-GFP and PfGatB-GFP do not localize to the mitochondrion in erythrocytic stages. The mitochondrion was labeled using MitoTracker Red, whereas PfGatA-GFP and PfGatB-GFP were detected via immunofluorescence assay using α-GFP antibody (green). Nucleic acid was stained with DAPI. Panels Ci and Cii show rings and early trophozoites, respectively, expressing PfGatA-GFP. In rings (C, panel i), the mitochondrion and apicoplast are clearly distinct organelles that enlarge and elongate during trophozoite development (C, panel ii). Panels Di and Dii show early and late schizonts, respectively. Nuclear division is underway in early schizonts (panel Di), and the apicoplast and mitochondrion are beginning to elongate. In late schizogony (panel Dii), daughter merozoites have formed, and the apicoplast (green) and the mitochondrion (red) have segregated to daughter merozoites.

FIGURE 4.

Integration and localization to the apicoplast of a second copy of GatA fused to a quadruple Myc tag (PbGatA-myc) in the P. berghei genome. A, 3.5-kb fragment of the 3′ end of the GatA gene without the stop codon was amplified from P. berghei ANKA genomic DNA and ligated upstream of the quadruple Myc tag in the b3D myc vector (18). Following linearization of the construct with PacI, it was transfected into P. berghei blood stage schizonts that were subsequently injected into mice. The vector contains a mutated T. gondii DHFR/TS gene as a pyrimethamine selectable marker (TgDHFR). Transgenic parasites were selected via pyrimethamine treatment and cloned by limiting dilution. B, PCR analysis of the integration site in a cloned PbGatA-myc transgenic (tr) parasite. Ethidium bromide-stained agarose gels showing the integration of the PbGatA-myc vector into the P. berghei genome. Only PbGatA-myc is positive in the 3′ and 5′ integration (int) tests, whereas both PbGatA-myc and wild type (wt) parasite genomic DNAs are positive for the PbGatA open reading frame test (ORF test). M indicates the size ladder. C, PbGatA-Myc apicoplast localization was monitored via immunofluorescence assay using an anti-Myc antibody (green), and the apicoplast was detected by staining with anti-ACP antibody (red). Nucleic acid was stained with DAPI (blue). PbGatA-Myc forms a characteristically small and round compartment early in the infection cycle (top row), which then elongates and develops into a complex and multiply branched form at the trophozoite stage prior to splitting into individual spots, one for each merozoite, in the schizont stage (bottom row). PfGatA-Myc is co-localized with ACP (α-Myc/α-ACP overlay), confirming localization to the plastid. D, PbGatA-Myc did not localize to the mitochondrion in erythrocytic stages. The mitochondrion was labeled using MitoTracker Red, and PbGatA-Myc was detected via immunofluorescence assay using an anti-Myc antibody (green). Nucleic acid was stained with DAPI. In the early stages of parasite development (top row), PbGatA-Myc and mitochondria are discrete single organelles. In the late trophozoite stages, the mitochondria and PbGatA-Myc are heavily branched but mostly distinct with a few points of overlapping signal (bottom row). Scale bar 1 μm.

FIGURE 5.

Attempted knock-out of the PbGatA gene via double-crossover recombination. Two fragments containing 5′- and 3′-UTRs of the PbGluRS gene, with ≈100 nucleotides of the start and end, respectively, of the PbGatA coding sequence were cloned into the B3D KO Red vector (38). After linearization with ApaI, the construct was transfected into P. berghei ANKA parasites (39). In three independent experiments, we were unable to delete the endogenous PbGatA gene by integrating the deletion construct via double-crossover recombination.

FIGURE 6.

PfGatAB purification and amidotransferase assay. A, purification of PfGatA assessed by 7.5% SDS-PAGE. Lanes 1 and 2, eluate from Ni-NTA column; lane 3, eluate from gel filtration chromatography. B, purification of the PfGatB assessed by 7.5% SDS-PAGE. Lanes 1–3, eluate from Ni-NTA column; lane 4, eluate from gel filtration purification. C, representative phosphorimage of the separation of Gln-[α-³²P]AMP, Glu-[α-³²P]AMP, and [α-³²P]AMP by PEI-cellulose TLC after PfGatAB-catalyzed amidotransferase reactions. The amidotransferase assays were performed as described under “Experimental Procedures” with the following modifications. Lanes 1 and 2 contained PfGatA, PfGatB, and either ³²P-labeled apicoplast tRNA^Gln or tRNA^Glu substrates in the presence of l-glutamate. No Glu-tRNA^Gln or Glu-tRNA^Glu was observed, implying that PfGatAB does not possess aminoacylation activity. Lanes 3 and 4 contained PfGatA, PfGatB, and either ³²P-labeled Glu-tRNA^Glu or Glu-tRNA^Gln in the presence of l-glutamate. No reaction was observed, implying that PfGatAB does not utilize l-Glu as a substrate. Lanes 5 and 6 contained PfGatA, PfGatB, and either ³²P-labeled Glu-tRNA^Glu or Glu-tRNA^Gln in the presence of l-glutamine. PfGatAB converted Glu-tRNA^Gln to Gln-tRNA^Gln but did not utilize Glu-tRNA^Glu as a substrate, indicating that PfGatAB has a high specificity for Gln-tRNA^Gln. D, representative phosphorimage of the effect of different amide donors on amidation activity. Lane 1 contained 50 nm each of PfGluRS, PfGatA, PfGatB, and ³²P-labeled apicoplast tRNA^Gln. Lane 2 contained 50 nm each of PfGluRS, PfGatA, PfGatB, ³²P-labeled apicoplast tRNA^Gln, and l-glutamate. Lanes 3 and 4 contained 50 nm each of PfGluRS, PfGatA, PfGatB, ³²P-labeled apicoplast tRNA^Gln, and l-glutamate, plus either l-glutamine or l-asparagine as amide donors. PfGatAB utilizes both l-glutamine and l-asparagine as amide donors.