Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

doi:10.1016/0014-4894(82)90094-7

Experimental Parasitology

Volume 53, Issue 1, February 1982, Pages 77-86

https://doi.org/10.1016/0014-4894(82)90094-7 Get rights and content

Abstract

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.

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Nanos gigantium humeris insidentes: old papers informing new research into Toxoplasma gondii
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As important as Pfefferkorn’s 1984 study is, it was just one contribution in a remarkable career. In addition to revealing a critical role for IFN-γ in mediating host responses to T. gondii infection, Pfefferkorn made key discoveries in nutrient salvaging pathways (Schwartzman and Pfefferkorn, 1982), and the delayed-death phenomenon observed with drugs that target the apicoplast (Pfefferkorn and Borotz, 1994), as well as establishing powerful genetic approaches that attracted many young researchers to our field (Pfefferkorn and Pfefferkorn, 1980). The recent passing of Elmer Pfefferkorn, and another giant in the field, Jack Remington, also remind us to reflect not only on their seminal research contributions, but also on the direct influence they both had on so many researchers who are continuing their scientific legacies today.
Since Nicolle, Manceaux and Splendore first described Toxoplasma gondii as a parasite of rodents and rabbits in the early 20th century, a diverse and vigorous research community has been built around studying this fascinating intracellular parasite. In addition to its importance as a pathogen of humans, livestock and wildlife, modern researchers are attracted to T. gondii as a facile experimental system to study many aspects of evolutionary biology, cellular biology, host-microbe interactions, and host immunity. For new researchers entering the field, the extensive literature describing the biology of the parasite, and the interactions with its host, can be daunting. In this review, we examine four foundational studies that describe various aspects of T. gondii biology, presenting a ‘journal club’-style analysis of each. We have chosen a paper that established the beguiling life cycle of the parasite (Hutchison et al., 1971), a paper that described key features of its cellular biology that the parasite shares with related organisms (Gustafson et al., 1954), a paper that characterised the origin of the unique compartment in which the parasite resides within host cells (Jones and Hirsch, 1972), and a paper that established a key mechanism in the host immune response to parasite infection (Pfefferkorn, 1984). These interesting and far-reaching studies set the stage for subsequent research into numerous facets of parasite biology. As well as providing new researchers with an entry point into the literature surrounding the parasite, revisiting these studies can remind us of the roots of our discipline, how far we have come, and the new directions in which we might head.
Contact and competition between mitochondria and microbes
2021, Current Opinion in Microbiology
Invading microbes occupy the host cytosol and take up nutrients on which host organelles are also dependent. Thus, host organelles are poised to interact with intracellular microbes. Despite the essential role of host mitochondria in cellular metabolic homeostasis and in mediating cellular responses to microbial infection, we know little of how these organelles interact with intracellular pathogens, and how such interactions affect disease pathogenesis. Here, we give an overview of the different classes of physical and metabolic interactions reported to occur between mitochondria and eukaryotic pathogens. Investigating the underlying molecular mechanisms and functions of such interactions will reveal novel aspects of infection biology.
Biochemistry and metabolism of Toxoplasma gondii: Purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa
2020, Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods
As obligate intracellular protozoa, Toxoplasma gondii and other Apicomplexa must acquire nutrients from their host cells for replication. Amongst the most important nutrients are nucleobases and nucleosides that form the essential building blocks for nucleic acids and molecules required for energy metabolism. There is a diverse repertoire of purine and pyrimidine pathways in the Apicomplexa. This diversity in metabolic pathways reflects the availability of essential nutrients in the particular host-cell niches occupied by the Apicomplexa, and the strategies used to generate purine and pyrimidine nucleotides. All Apicomplexa are unable to make purines de novo and must salvage purines from their hosts. The ability to make pyrimidines de novo and to salvage pyrimidines from the host varies between the different apicomplexan parasites. This chapter reviews our current knowledge of purine and pyrimidine metabolic pathways for three major Apicomplexa of importance in human disease: Plasmodium species, Cryptosporidium species, and T. gondii.
Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine
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Initial velocity and product inhibition studies of Toxoplasma gondii adenosine kinase (TgAK, EC 2.7.1.20) demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate–enzyme–co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were K_Ado = 0.002 ± 0.0002 mM, K_ATP = 0.05 ± 0.008 mM, and V_max = 920 ± 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5′-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.
Influence of infection by Toxoplasma gondii on purine levels and E-ADA activity in the brain of mice experimentally infected mice
2014, Experimental Parasitology
Citation Excerpt :
Studies in cell culture show that when there is a largest decline in the percent of viable astrocytes during ischemia, occurs a concomitant reduction in ATP and ADP levels (Yu et al., 2002). Our first hypothesis lies on the fact that although T. gondii can synthesize pyrimidines de novo, it lacks the ability to synthesize purines, including adenosine (Schwartzman and Pfefferkorn, 1982). The parasite then, uses the host purines, especially when CD73 (5′nucleotidase) mediates the final rate-limiting step in the enzymatic chain that catalyzes the dephosphorylation of extracellular AMP to adenosine from ATP.
The aim of this study was to assess the purine levels and E-ADA activity in the brain of mice (BALB/c) experimentally infected with Toxoplasma gondii. In experiment I (n = 24) the mice were infected with RH strain of T. gondii, while in experiment II (n = 36) they were infected with strain ME-49 of T. gondii. Our results showed that, for RH strain (acute phase), an increase in both periods in the levels of ATP, ADP, AMP, adenosine, hypoxanthine, xanthine (only on day 6 PI) and uric acid (only on day 6 PI). By the other hand, the RH strain led, on days 4 and 6 PI, to a reduction in the concentration of inosine. ME-49, a cystogenic strain, showed some differences in acute and chronic phase, since on day 6 PI the levels of ATP and ADP were increased, while on day 30 these same nucleotides were reduced. On day 60 PI, ME-49 induced a reduction in the levels of ATP, ADP, AMP, adenosine, inosine and xanthine, while uric acid was increased. A decrease of E-ADA activity was observed in brain on days 4 and 6 PI (RH), and 30 PI (ME-49); however on day 60 PI E-ADA activity was increased for infection by ME-49 strain. Therefore, it was possible to conclude that infection with T. gondii changes the purine levels and the activity of E-ADA in brain, which may be associated with neurological signs commonly observed in this disease.
Exploitation of auxotrophies and metabolic defects in Toxoplasma as therapeutic approaches
2014, International Journal for Parasitology
Like any obligate intracellular pathogen, the parasite Toxoplasma gondii has lost its capacity for living independently of another organism. Toxoplasma lacks many genes that encode for entire metabolic pathways and has, in return, expanded genes that promote nutrient scavenging to meet its basic metabolic requirements. Although sequestrated in a parasitophorous vacuole and thus insulated from the nutrient-rich host cytosol and organelles by a membrane, T. gondii has evolved efficient strategies to acquire essential metabolites from mammalian cells. This review explores the natural auxotrophies and nutrient scavenging activities of the parasite, emphasising unique transport systems and salvage pathways. We describe the mechanisms deployed by Toxoplasma to modify its parasitophorous vacuole to gain access to host cytosolic molecules and to hijack host organelles to retrieve their nutrient content. From a therapeutic perspective, we survey the different possibilities to starve T. gondii by nutrient depletion or disruption of salvage pathways.

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¹: Present address: Clinical Laboratories, Department of Pathology, Medical Center, University of Virginia P.O. Box 168 Charlottesville, Virginia 22908, U.S.A.

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Experimental Parasitology

Abstract

References (25)

Some studies on the DNA of Plasmodium knowlesi

Detailed purine salvage metabolism in and outside the free malarial parasite

Experimental Parasitology

Toxoplasma gondii: Isolation and preliminary characterization of temperature-sensitive mutants

Experimental Parasitology

Toxoplasma gondii: Specific labeling of nucleic acids of intracellular parasites in Lesch-Nyhan cells

Experimental Parasitology

Toxoplasma gondii: Characterization of a mutant resistant to 5-fluorodeoxyuridine

Experimental Parasitology

Plaque assay of Toxoplasma on monolayers of chick embryo fibroblasts

The selection of Chinese hamster cells deficient in oxidative energy metabolism

Somatic Cell Genetics

Nutritional needs of mammalian cells in tissue culture

Science

Incorporation of radioactive precursors into DNA and RNA of Plasmodium knowlesi in vitro

Journal of Protozoology

Purine base and nucleoside uptake in Plasmodium berghei and host erythrocytes

Journal of Parasitology

The interaction between Toxoplasma gondii and mammalian cells. I. Mechanism of entry and intracellular fate of the parasite

Journal of Experimental Medicine

The uptake and incorporation of nucleosides into normal erythrocytes and erythrocytes containing Plasmodium berghei

Parasitology

Cited by (105)

Nanos gigantium humeris insidentes: old papers informing new research into Toxoplasma gondii

Contact and competition between mitochondria and microbes

Biochemistry and metabolism of Toxoplasma gondii: Purine and pyrimidine acquisition in Toxoplasma gondii and other Apicomplexa

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

Influence of infection by Toxoplasma gondii on purine levels and E-ADA activity in the brain of mice experimentally infected mice

Exploitation of auxotrophies and metabolic defects in Toxoplasma as therapeutic approaches