Mirtha M. Flawiá

A Neurospora crassa morphological mutant showing reduced adenylate cyclase activity.

Summary A Neurospora crassa morphological mutation at the crisp locus is related to a low specific activity of adenylate cyclase. This observation was made in two different strains: FGSC No. 329 and FGSC No. 326, both carrying the crisp mutation (allele N. B 123). Crosses of these strains to others having the wild type allele yield isolates with the crisp phenotype all of them having low adenylate cyclase specific activity in crude extracts. The other phenotypes obtained showed wild type levels of specific activity. Addition of 3′, 5′ dibutyryl cyclic AMP to the FGSC No. 329 cultures increased four-fold mycelial growth rate and stimulated the formation of aerial hyphae. Simultaneously, uniform conidiation on the surface of the slant was inhibited by the cyclic AMP derivative.

Arginine kinase overexpression improves Trypanosoma cruzi survival capability

Arginine kinase catalyzes the reversible transphosphorylation between adenosine diphosphate (ADP) and phosphoarginine, which is involved in temporal and spatial adenosine triphosphate (ATP) buffering. Here we demonstrate that the homologous overexpression of the Trypanosoma cruzi arginine kinase improves the ability of the transfectant cells to grow and resist nutritional and pH stress conditions. The stable transfected parasites showed an increased cell density since day 10 of culture, when the carbon sources became scarce, which resulted 2.5‐fold higher than the control group on day 28. Additional stress conditions were also tested. We propose that arginine kinase is involved in the adaptation of the parasite to environmental changes.

L‐Arginine Uptake and L‐Phosphoarginine Synthesis In Trypanosoma Cruzi

ABSTRACT A very specific L‐arginine transporter showing high affinity has been characterized in Trypanosoma cruzi epimastigotes. Uptake was found to be dependent on L‐arginine concentration and it was saturable. Values for maximum velocity and Km ranged between 48.1‐57.5 pmol·min‐1 per 3 times 10‐ cells and between 4.2‐5.5 μM, respectively. the calculated activation energy and Q10 were 31.1 KJ·mol‐1, and 1.7, respectively. Uptake velocity significantly increased when cells were preincubated in the absence of L‐arginine, Cells retained the labeled amino acid independently of the presence or absence of exogenous L‐arginine. the specificity of L‐arginine uptake was demonstrated by competition assays in the presence of 80‐fold molar excess of natural amino acids and several L‐arginine derivatives. the highest levels of inhibition were caused by L‐homoarginine, D‐arginine, L‐canavanine, L‐ornithine, and L‐citrulline. L‐arginine uptake by T. cruzi epimastigotes was not affected by the presence of potassium or sodium ions in the incubation mixture or by pH changes in the range between 5.5‐8.5. the major product of L‐arginine uptake was characterized as phosphoarginine. Moreover, arginine kinase activity was detected in soluble extracts from T. cruzi epimastigotes.

Arginine Kinase: A Common Feature for Management of Energy Reserves in African and American Flagellated Trypanosomatids

Abstract This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 μmol min−1mg−1. Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.

Arginine kinase of the flagellate protozoa Trypanosoma cruzi: Regulation of its expression and catalytic activity

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas’ disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme‐specific activity and enzyme protein was observed. Arginine kinase‐specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post‐transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

The initiation of glycogen biosynthesis in Escherichia coli

Previous work from this laboratory has shown that a protein acts as the initial acceptor of glucose from UDPglucose for glycogen biosynthesis in rat liver [ 1,2]. Because of the different solubility of protein and glycogen in trichloroacetic acid, it has been possible to determine differentially the incorporation of radioactivity from labelled sugar nucleotide into protein or into glycogen. Based on results obtained with rat liver preparations [3], we have proposed the participation of two enzymes and an acceptor protein in the initiation of glycogen biosynthesis. We have applied the same approach to the study of glycogen initiation in bacteria. In this paper we report the isolation from Escherichia cofi of an enzyme complex which synthesizes a 1,4-ol-glucoprotein not only from ADPglucose, which is considered to be the specific donor in bacterial glycogen biosynthesis but also from UDPglucose.

Arginine metabolism in Trypanosoma cruzi is coupled to parasite stage and replication.

L‐Arginine plays an essential role in the energetic metabolism of Trypanosoma cruzi. In this work we propose a relationship between L‐arginine uptake, arginine kinase activity and the parasite replication ability. In epimastigote cultures L‐arginine uptake decreases continuously accompanying a cell replication rate reduction. The use of conditioned or fresh medium mimics uptake variations. Interestingly, in non‐replicative trypomastigote cells, L‐arginine uptake was undetectable. The association between L‐arginine uptake and cell replication was demonstrated using the antimitotic agent hydroxyurea. Arginine kinase, the enzyme responsible for phosphoarginine and ATP synthesis, also shows a differential activity in epimastigote and trypomastigote parasite stages.

Signal transduction mechanisms in Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, is an adequate model for studies on the evolution of signal transduction pathways. These pathways involve molecular entities such as membrane receptors, transduction G proteins, protein kinases and second messengers (Ca(2+), cyclic AMP, cyclic GMP, nitric oxide). In this article, Mirtha M. Flawiá, María T. Téllez-Iñón and Héctor N. Torres describe the studies performed on T. cruzi transduction pathways and their role in the control of metacyclogenesis and cell motility.

A novel calcium-stimulated adenylyl cyclase from Trypanosoma cruzi, which interacts with the structural flagellar protein paraflagellar rod.

Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member ofT. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiaemutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (Km = 406 μm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn2+and the P-site inhibitor 2′-deoxyadenosine 3′-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca2+ which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.

A Trypanosoma cruzi phosphatidylinositol 3-kinase (TcVps34) is involved in osmoregulation and receptor-mediated endocytosis.

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.