Kisiala, Anna B
The effect of cytokinins on the metabolite secretome of Giardia intestinalis during trophozoite growth, nutrient deprivation, and encystation
Giardia intestinalis is the causative agent of a diarrheal disease in mammals, but the mechanisms of disease pathogenesis are unclear. While proteins secreted by Giardia affect the host cells, the potential of hormone secretion has not been investigated to date. Cytokinins (CKs) are classified as phytohormones, but little is known about their role beyond plants. Mass spectrometry-based intracellular analysis revealed CKs typical of tRNA degradation, and extracellular analysis showed CK-riboside scavenging by Giardia with concurrent secretion of CK-free bases. Metabolomics profiling of culture supernatants showed similar trends where nucleosides were up taken, and nucleobases were secreted. The dynamics of amino acids, nucleosides and nucleobases were altered by CK-supplementation during encystation, along with inhibition of encystation. In summary, this is the first study to report CK synthesis and metabolism by Giardia along with the effects of CKs on the metabolite secretome of Giardia, while establishing a link between CK and nucleoside metabolism.
Author Keywords: Cytokinins, Giardia, mass spectrometry, metabolomics, parasite, secretome
The first confirmation that Insects synthesize Cytokinins: Cytokinin metabolite and gene expression profiling following functional manipulations of tRNA IPT genes in Drosophila melanogaster
Using Drosophila deficiency (Df) and Over Expression (OE) (GAL4/UAS>dCas9-VPR; sgRNA) gene systems, it was demonstrated that Dmel_CG31381 and Dmel_CG11089 are functional tRNA isopentenyltransferase (EC 2.5.1.8) genes (tRNA IPT1 and IPT2) critical to the first committed step in insect cytokinin biosynthesis. IPT Df mutants showed significant decreases in total CK levels and IPT1/IPT2 transcript levels compared to parent lines. IPT OE mutants showed significant increases in total CK levels and IPT1/IPT2 transcript levels compared to parent lines. Further, endogenous CK analyte levels and qPCR relative fold gene expression of Dmel_CG31381 and Dmel_CG11089 (tRNA IPT1 and IPT2) genes demonstrated expression patterns with functional confirmation corresponding to the predicted IPT mutant variants. The functional confirmation of tRNA IPT1 and IPT2 as the first committed step was further supported by the bioinformatic detection of putative gene homologs to corroborate seven remaining enzyme transcripts supporting the novel description of a CK biosynthesis pathway in insects.
Author Keywords: Cytokinin Biosynthesis, Drosophila, gene expression, Insect Gall, mass spectrometry, tRNA IPT
The effect of cytokinins on the metabolite secretome of Giardia intestinalis during trophozoite growth, nutrient deprivation, and encystation
Giardia intestinalis is the causative agent of a diarrheal disease in mammals, but the mechanisms of disease pathogenesis are unclear. While proteins secreted by Giardia affect the host cells, the potential of hormone secretion has not been investigated to date. Cytokinins (CKs) are classified as phytohormones, but little is known about their role beyond plants. Mass spectrometry-based intracellular analysis revealed CKs typical of tRNA degradation, and extracellular analysis showed CK-riboside scavenging by Giardia with concurrent secretion of CK-free bases. Metabolomics profiling of culture supernatants showed similar trends where nucleosides were up taken, and nucleobases were secreted. The dynamics of amino acids, nucleosides and nucleobases were altered by CK-supplementation during encystation, along with inhibition of encystation. In summary, this is the first study to report CK synthesis and metabolism by Giardia along with the effects of CKs on the metabolite secretome of Giardia, while establishing a link between CK and nucleoside metabolism.
Author Keywords: Cytokinins, Giardia, mass spectrometry, metabolomics, parasite, secretome