Biology subjects

Baptista, C. G.

Publications and source records attributed to Baptista, C. G..

5 recordsLinked to original sources

bioRxiv2024

Elucidating the Transport Mechanisms and Metabolic Roles of Serine, Threonine, and Glycine in Trypanosoma cruzi

O_SCPLOWLC_SCPLOW-Serine (O_SCPLOWLC_SCPLOW-Ser) and O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThreonine (O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr) have versatile roles in metabolism. In addition to their use in protein synthesis, these amino acids participate in the biosynthesis pathways of other amino acids and even phospholipids. Furthermore, O_SCPLOWLC_SCPLOW-Ser and O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr can be substrates for a Ser/Thr dehydratase (Ser/ThrDH), resulting in pyruvate (Pyr) and 2-oxobutyrate, respectively, thus being amino acids with anaplerotic potential. Trypanosoma cruzi, the etiological agent of Chagas disease, uses amino acids in several biological processes: metacyclogenesis, infection, resistance to nutritional and oxidative stress, osmotic control, etc. In this study, we investigated the import and metabolism of O_SCPLOWLC_SCPLOW-Ser, O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr, and Gly in T. cruzi. Our results demonstrate that these amino acids are transported from the extracellular environment into T. cruzi cells through a saturable transport system that fits the Michaelis-Menten model. Our results show that O_SCPLOWLC_SCPLOW-Ser and O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr can sustain epimastigote (Epi) cell viability under nutritional stress (NS) conditions and can stimulate oxygen consumption to maintain intracellular ATP levels. Additionally, our findings indicate that O_SCPLOWLC_SCPLOW-Ser plays a role in establishing the mitochondrial membrane potential ({Delta}{Psi}m) in T. cruzi. O_SCPLOWLC_SCPLOW-Ser is also involved in energy metabolism via the Ser-Pyr pathway, which stimulates the production and subsequent excretion of acetate and alanine. Our results demonstrate the importance of O_SCPLOWLC_SCPLOW-Ser and O_SCPLOWLC_SCPLOW-Thr in the energy metabolism of T. cruzi and provide new insights into the metabolic adaptations of this parasite during its life cycle.

biochemistry↗

bioRxiv2023

Toxoplasma gondii F-Box Protein L2 Silences Feline-Restricted Genes Necessary for Sexual Commitment

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages requires substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct nuclear sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNA seq data show that TgFBXL2 conditional depletion induces the expression of genes necessary for sexual commitment. We suggest that TgFBXL2 is a latent guardian of sexual stage development in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of sexual development. AUTHOR SUMMARYToxoplasma gondii is a protozoan parasite that replicates sexually in felids and asexually in nearly all other mammals with each life stage having a specific transcriptional profile. When life stage specific transcription is not properly controlled, the parasite dies and therefore its important to understand what inhibits expression of sexual stage genes during asexual growth and vice versa. Here we identify a ubiquitin E3 ligase complex that inhibits sexual stage gene expression during asexual growth.

microbiology↗

bioRxiv2023

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that TgCPDH potentially functions as an alternative enzyme to perform the same reaction as CPOX under low oxygen conditions. In this study, we demonstrated that TgCPDH exhibits coproporphyrinogen dehydrogenase activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wildtype and heme-deficient parasites did not alter their intracellular growth under both ambient and low oxygen conditions. This research marks the first report of a coproporphyrinogen dehydrogenase-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings suggest that it does not directly affect intracellular infection or the pathogenesis of Toxoplasma parasites. IMPORTANCEToxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its lifecycle, these parasites must adapt to varying environmental conditions, including situations with low oxygen levels. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase, suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, coproporphyrinogen dehydrogenase has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying coproporphyrinogen dehydrogenase activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

microbiology↗

bioRxiv2023

Identification of Toxoplasma Calcium Dependent Protein Kinase 3 as a Stress-Activated Elongation Factor 2 Kinase

Toxoplasma gondii is an obligate intracellular parasite whose tachyzoite form causes disease via a lytic growth cycle. Its metabolic and cellular pathways are primarily designed to ensure parasite survival within a host cell. But during its lytic cycle tachyzoites are exposed to the extracellular milieu and prolonged exposure requires activation of stress response pathways that include reprogramming the parasite proteome. Regulation of protein synthesis is therefore important for extracellular survival. We previously reported that in extracellularly stressed parasites that the elongation phase of protein synthesis is negatively regulated by the Toxoplasma oxygen sensing protein, PhyB. PhyB acts by promoting the activity of elongation factor eEF2, which is a GTPase that catalyzes the transfer of the peptidyl-tRNA from the A site to the P site of the ribosome. In the absence of PhyB, eEF2 is hyper-phosphorylated, which inhibits eEF2 from interacting with the ribosome. eEF2 kinases are atypical calcium-dependent kinases and BLAST analyses revealed the parasite kinase, CDPK3, as the most highly homologous to the Saccharomyces cerevisiae eEF2 kinase, RCK2. In parasites exposed to extracellular stress, loss of CDPK3 leads to decreased eEF2 phosphorylation and enhanced rates of elongation. Furthermore, co-immunoprecipitation studies revealed that CDPK3 and eEF2 interact in stressed parasites. Since CDPK3 and eEF2 normally localize to the plasma membrane and cytosol, respectively, we investigated how the two can interact. We report that under stress conditions that CDPK3 is not N-myristoylated likely leading to its cytoplasmic localization. In summary, we have identified a novel function for CDPK3 as the first protozoan extracellular-stress induced eEF2 kinase. IMPORTANCEHere, we identify the first protozoan kinase that phosphorylate elongation factor 2 and demonstrate that it is part of an integrated stress response.

microbiology↗

bioRxiv2019

Toxoplasma F-Box Protein 1 is Required for Daughter Cell Scaffold Function During Parasite Replication

By binding to the adaptor protein SKP1 and serving as substrate receptors for the Skp1, Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but occurred before segregation of the centrocone, which connects the spindle pole to the centrosomes. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.\n\nAUTHOR SUMMARYToxoplasma gondii is a protozoan parasite that can cause devastating and life-threatening disease in immunocompromised patients and in fetuses. Its replication is important to study because parasite growth is responsible for the pathology that develops in toxoplasmosis patients. The parasite replicates by a unique process named endodyogeny in which two daughter parasites develop within the mother cell. Early during this process the parasite creates a structure called the Daughter Cell Scaffold whose function is to mark the site from which daughter parasites will emerge. Here, we report the identification of one of the first proteins recruited to the Daughter Cell Scaffold and its importance in executing its function.

microbiology↗