Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii

Asai, Takashi; O'Sullivan, W.J.; Kobayashi, Masashi; Gero, Annette M.; Yokogawa, M.; Tatibana, Masamiti

doi:10.1016/0166-6851(83)90037-3

Cited by 63 publications

(31 citation statements)

References 19 publications

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“…As reported previously, we and others have identified the gene encoding nucleoside triphosphate (NTPase), 1 a very abundant protein secreted by T. gondii shortly after invasion of host cells (2)(3)(4)(5). The 63-kDa NTPase isoforms are encoded by two genes, NTP1 and NTP3, which share more than 97% sequence identity (2).…”

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confidence: 92%

“…The 63-kDa NTPase isoforms are encoded by two genes, NTP1 and NTP3, which share more than 97% sequence identity (2). When activated in vitro by dithiols, NTPase is one of the most potent apyrases known to date, capable of sequentially degrading ATP to ADP and AMP (4). Because T. gondii is a purine auxotroph, it is thought that NTPase may participate in purine salvage (2,6,7).…”

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confidence: 99%

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Targeted Reduction of Nucleoside Triphosphate Hydrolase by Antisense RNA Inhibits Toxoplasma gondii Proliferation

Nakaar

Samuel

Ngo

et al. 1999

Journal of Biological Chemistry

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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 hypoxanthinexanthine-guanine phosphoribosyl transferase (HXG-PRT) expression by a chimeric antisense HXGPRT RNAribozyme 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.

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confidence: 92%

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confidence: 99%

Targeted Reduction of Nucleoside Triphosphate Hydrolase by Antisense RNA Inhibits Toxoplasma gondii Proliferation

Nakaar

Samuel

Ngo

et al. 1999

Journal of Biological Chemistry

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“…The enzyme contains 15 cysteine residues, and in vitro, requires dithiols to be enzymatically active. Dithiothreitol (DTT) will activate the enzyme, whereas monothiols such as glutathione and cysteine are ineffective (5). Thioredoxin is reported to activate the protein (11); however, host cell thioredoxin is presumably excluded from the parasitophorous vacuole by the PVM, and it is not known whether Toxoplasma secretes a physiologic dithiol.…”

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confidence: 99%

“…Indeed, theoretical considerations suggest that it cannot be fully active. The NTPase constitutes 1-2% of total parasite protein (5); based on in vitro activity levels, we have calculated that the fully activated enzyme secreted by a single parasite could degrade the entire host ATP pool in a matter of minutes, assuming free access to ATP via the PVM pore. These considerations suggest that the enzymatic activity of the NTPase must be tightly regulated and raise the question * The costs of publication of this article were defrayed in part by the payment of page charges.…”

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confidence: 99%

Induced Activation of the Toxoplasma gondiiNucleoside Triphosphate Hydrolase Leads to Depletion of Host Cell ATP Levels and Rapid Exit of Intracellular Parasites from Infected Cells

Silverman¹,

Qi²,

Riehl

et al. 1998

Journal of Biological Chemistry

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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.

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“…In the absence of any pyrimidine salvage pathway, T. gondii still possesses a complete six-step pathway for the de novo biosynthesis of UMP, the precursor molecule of all essential pyrimidines (1,14,34,38). Insertional disruption of the first step of the biosynthetic pathway, encoded by the carbamoyl phosphate synthetase II (CPSII) gene, produced a severe uracil auxotrophy exemplified by the cps1-1 strain of T. gondii, which was incapable of de novo pyrimidine synthesis (13,16).…”

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confidence: 99%

Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to Toxoplasma gondii

Fox¹,

Bzik²

2010

Infect Immun

125

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The orotidine-5-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting ⌬OMPDC, ⌬UP, and ⌬OMPDC ⌬UP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The ⌬UP knockout strain was replication competent and virulent. In contrast, the ⌬OMPDC and ⌬OMPDC ⌬UP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the ⌬OMPDC strain but not the ⌬OMPDC ⌬UP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the ⌬OMPDC and ⌬OMPDC ⌬UP knockout strains exhibited extreme attenuation in murine virulence (ϳ8 logs). Genetic complementation of the ⌬OMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated ⌬OMPDC strain or the ⌬OMPDC ⌬UP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting ⌬OMPDC and ⌬OMPDC ⌬UP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered.

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Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii

Cited by 63 publications

References 19 publications

Targeted Reduction of Nucleoside Triphosphate Hydrolase by Antisense RNA Inhibits Toxoplasma gondii Proliferation

Targeted Reduction of Nucleoside Triphosphate Hydrolase by Antisense RNA Inhibits Toxoplasma gondii Proliferation

Induced Activation of the Toxoplasma gondiiNucleoside Triphosphate Hydrolase Leads to Depletion of Host Cell ATP Levels and Rapid Exit of Intracellular Parasites from Infected Cells

Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to Toxoplasma gondii

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