Selection based on the expression of antisense hypoxanthine-xanthine-guanine-phosphoribosyltransferase RNA in Toxoplasma gondii.

We have previously shown that an antisense RNA strategy can be used to inhibit the expression of hypoxanthine-xanthine-guanine-phosphoribosyltransferase (HXGPRT) in Toxoplasma gondii [Nakaar et al., J. Biol. Chem. 1999;274:5083-5087]. Here, we report that parasites rendered deficient in HXGPRT by antisense RNA are resistant to high doses of 6-thioxanthine (6-TX). We have exploited this finding to develop a selection procedure. In this scheme, parasites transfected with a chimeric construct harboring the bacterial chloramphenicol acetyl transferase (CAT) reporter gene linked to antisense HXGPRT gene were selected in 6-TX to inhibit the growth of tachyzoites expressing endogenous HXGPRT. Concomitant with a reduction in HXGPRT levels by antisense RNA, 6-TX(R) parasites displayed reporter CAT activity. These data indicate that transfection of antisense HXGPRT gene provides a means to select for parasites expressing foreign or altered genes in T. gondii. These findings also suggest, in principle, that antisense RNA can be used as a strategy to generate selectable markers employing genes that encode enzymes with known subversive substrates.

Targeted reduction of nucleoside triphosphate hydrolase by antisense RNA inhibits Toxoplasma gondii proliferation.

Nucleoside triphosphate hydrolase (NTPase) is a very abundant protein secreted by the obligate intracellular parasite Toxoplasma gondii shortly after invasion of the host cell. When activated by dithiols, NTPase is one of the most potent apyrases known to date, but its physiological function remains unknown. The genes encoding NTPase have been cloned (Bermudes, D., Peck, K. R., Afifi-Afifi, M., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260). We have recently shown that the enzyme is tightly controlled within the vacuolar space and may influence parasite exit from the host cell (Silverman, J. A., Qi, H., Riehl, A., Beckers, C., Nakaar, V., and Joiner, K. A (1998) J. Biol. Chem. 273, 12352-12359). In the present study, we have generated an antisense NTP RNA construct in which the 3'-untranslated region is replaced by a hammerhead ribozyme. The constitutive synthesis of the chimeric antisense RNA-ribozyme construct in parasites that were stably transfected with this construct resulted in a dramatic reduction in the steady-state levels of NTPase. This inhibition was accompanied by a decrease in the capacity of the parasites to replicate. The reduction in parasite proliferation was due to a specific effect of antisense NTP RNA, since a drastic inhibition of hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) expression by a chimeric antisense HXGPRT RNA-ribozyme construct did not alter NTPase expression nor compromise parasite replication. These data implicate NTPase in an essential parasite function and suggest that NTPase may have more than one function in vivo. These results also establish that it is possible to study gene function in apicomplexan parasites using antisense RNA coupled to ribozymes.

Upstream elements required for expression of nucleoside triphosphate hydrolase genes of Toxoplasma gondii.

Nucleoside triphosphate hydrolase is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii. The protein has apyrase activity, degrading ATP to the di- and mono-phosphate forms. Because T. gondii is incapable of de novo synthesis of purines, it is postulated that NTPase may be used by the parasite to salvage purines from the host cell for survival and replication. To elucidate the molecular mechanisms of NTP gene expression, we isolated from the virulent RH strain of T. gondii the putative promoter region of three tandemly repeated NTP genes (NTP1, 2, 3). Using deletion constructs linked to the chloramphenicol acetyl transferase (CAT) reporter gene, we defined an active promoter within the first 220 bp. Sequence analysis of this region reveals the lack of a TATA box, but the promoter region is associated with a sequence which resembles an initiator element (Inr) in the NTP1 and NTP3 genes. This sequence which is similar to other Inrs known to regulate the expression of a wide variety of RNA polymerase II genes, is required for NTP expression. The NTP3 promoter contains sufficient information for developmentally regulated expression of CAT activity when the actively replicating stage tachyzoite differentiates into the dormant bradyzoite form.

Induced activation of the Toxoplasma gondii nucleoside triphosphate hydrolase leads to depletion of host cell ATP levels and rapid exit of intracellular parasites from infected cells.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase. The protein is synthesized in large amounts and transported through the secretory pathway of the parasite and into the vacuolar space in an oxidized and thereby enzymatically inactive form. Complete activation of the purified enzyme is known to require dithiols (e.g. DTT); subcellular fractionation demonstrates that little if any (<5%) of the enzyme in the vacuolar space is active in the absence of DTT. Both native and epitope-tagged nucleoside triphosphate hydrolase (NTPase) were partially activated during immunoprecipitation, precluding precise assessment of enzyme activity in the vacuolar space but suggesting that protein-protein interactions may trigger activation. When infected cells were treated with DTT, the NTPase was activated in a dose-response fashion, as assessed by migration on SDS-polyacrylamide gel electrophoresis and by an increase in enzymatic activity. After activation, enzyme activity decreased with time in the presence of DTT; this inactivation was slowed by the presence of excess ATP. A rapid fall in host cell ATP was accompanied by an abrupt exit of parasites from cells. These results demonstrate that the oxidation/reduction status of the NTPase, the only parasite dense granule protein that contains disulfide bonds, is tightly controlled within the vacuolar space and may influence parasite exit from cells.

Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase.

The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene family to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner KA. J Biol Chem 1994;269:29252-29260. Previous work also identified two isoforms of the enzyme in the virulent RH strain, and demonstrated that internal fragments of the genes encoding these isoforms were found differentially in virulent versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayashi H, Tsutomu T, J Biol Chem 1995;270:11391-11397). We now show that the NTPase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain lacks the gene for NTPase 3, explaining the absence of expression. Despite the fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid level, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cleaves predominantly nucleotide triphosphates. Furthermore, native and recombinant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agarose, further distinguishing the two isoforms. Using chimeras between the NTP1 and NTP3 genes, we show that a block of twelve residues at the C-terminus dictates substrate specificity. These residues lie outside the regions conserved among other E-ATPases, and therefore provide new insight into substrate recognition by this class of enzymes.

Structure of the Trypanosoma brucei U6 snRNA gene promoter.

Transcription in vivo of small nuclear and cytoplasmic RNA genes of Trypanosoma brucei was previously shown to require the A and B blocks of a divergently transcribed tRNA or tRNA-like gene located approximately 100 nucleotides (nt) upstream. To understand the functioning of these transcription units, we have used the U6 snRNA/tRNA(Thr) genes as a model system. Saturation mutagenesis revealed that for transcription in vivo three elements are essential and sufficient. In addition to the previously described A and B boxes, sequences in the U6 coding region close to the 5' end participate in positioning RNA polymerase III at the start site, and thus constitute a third promoter element. We further showed that the function of the upstream A box, but not the B box, is strictly dependent upon its distance to the U6 gene internal control region. Using our recently developed transcription extract we further demonstrated that in vitro U6 transcription requires only the intragenic sequences and the upstream A box of the tRNA(Thr) gene. This apparent discrepancy between the in vivo and in vitro requirements is highly reminiscent of U6 snRNA gene transcription in the yeast Saccharomyces cerevisiae, and suggests the possibility that similar to the yeast system the B block of the trypanosome U6 snRNA gene promoter might be involved in chromatin organization.

Accurate transcription of the Trypanosoma brucei U2 small nuclear RNA gene in a homologous extract.

In vitro transcription systems are a classic means to dissect mechanisms of gene expression at the molecular level. To begin an analysis of the biochemistry of gene expression in trypanosomes, we established an in vitro transcription system from cultured insect forms of Trypanosoma brucei. As a model we used the U2 snRNA gene which in vivo is transcribed by an RNA polymerase with characteristics of animal RNA polymerase III. To obtain maximum sensitivity in our assay, we adapted the so-called G-less cassette approach to the U2 snRNA gene promoter. Since an intragenic control region is required for accurate expression in vivo, we generated a series of mutations to substitute all guanosine residues in the intragenic control region. These mutants were shown to retain full transcriptional activity in vivo after transient expression in insect-form trypanosomes. In a cell-free extract, synthesis of the U2 G-less cassette RNA is correctly initiated, is mediated by RNA polymerase III as determined by RNA polymerase inhibitor studies, and is dependent on the integrity of the upstream B box element.

Upstream tRNA genes are essential for expression of small nuclear and cytoplasmic RNA genes in trypanosomes.

An interesting feature of trypanosome genome organization involves genes transcribed by RNA polymerase III. The U6 small nuclear RNA (snRNA), U-snRNA B (the U3 snRNA homolog), and 7SL RNA genes are closely linked with different, divergently oriented tRNA genes. To test the hypothesis that this association is of functional significance, we generated deletion and block substitution mutants of all three small RNA genes and monitored their effects by transient expression in cultured insect-form cells of Trypanosoma brucei. In each case, two extragenic regulatory elements were mapped to the A and B boxes of the respective companion tRNA gene. In addition, the tRNA(Thr) gene, which is upstream of the U6 snRNA gene, was shown by two different tests to be expressed in T. brucei cells, thus confirming its identity as a gene. This association between tRNA and small RNA genes appears to be a general phenomenon in the family Trypanosomatidae, since it is also observed at the U6 snRNA loci in Leishmania pifanoi and Crithidia fasciculata and at the 7SL RNA locus in L. pifanoi. We propose that the A- and B-box elements of small RNA-associated tRNA genes serve a dual role as intragenic promoter elements for the respective tRNA genes and as extragenic regulatory elements for the linked small RNA genes. The possible role of tRNA genes in regulating small RNA gene transcription is discussed.