Abstract
Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.
Highlights
Parasites display metabolic peculiarities that help them adapt to different environments during their life cycles and take advantage of the host’s resources
We show that the enzyme glutamine synthetase contributes to the management of excess ammonium and uses it to form the amino acid glutamine
The parasite invades mammalian host cells and transiently becomes enclosed in a tight vacuole, where it differentiates into the amastigote, an amino acid consumer stage
Summary
Parasites display metabolic peculiarities that help them adapt to different environments during their life cycles and take advantage of the host’s resources. Trypanosoma cruzi, the causative agent of Chagas disease, is a digenetic protozoan that transitions among different environments in its vertebrate and invertebrate hosts during its life cycle, alternating between nonreplicative and replicative stages [1]. Epimastigotes (E), which are the replicative forms in the insect vector, colonize the digestive tube and differentiate into non-dividing infective metacyclic trypomastigotes (MT) in its terminal portion [2]. MT must invade the mammalian host cells through an energy-dependent mechanism to be able to differentiate into replicative stages and establish infection [3,4]. After a variable number of cell divisions, A differentiate into a transient replicative form called intracellular epimastigotes (IE), which differentiate into cell-derived trypomastigotes (CDT) [11]. CDT lyse the infected cells, and once they burst, the CDT have two fates: i. to infect neighbor cells; and ii. to reach the bloodstream, from which they can reach and infect remote tissues, or if a triatomine makes a bloodmeal while the CDT are circulating, they can infect a new insect, which will transmit the parasite into new mammalian hosts [6]
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