Yee, Janet

The parasitic protist Giardia intestinalis has five heme proteins: a flavohemoglobin and several isotypes of cytochrome b5. While the flavohemoglobin has a role in counteracting nitric oxide, the functions of the cytochromes (gCYTb5s) are unknown. In this study, the protein level and cellular localization of three gCYTB5 isotypes (gCYTb5-I, II and III) and flavohemoglobin were examined in Giardia trophozoites exposed to three nitrosative stressors at two different concentrations: nitrite (20 mM, 0.5 mM); GSNO (2 mM, 0.25 mM) and DETA-NONOate (2 mM, 0.05 mM). An increase in protein levels was observed for gCYTb5-II with all stressors at both concentrations. However, the effects of these nitrosative stressors on gCYTb5-I and III were inconclusive due to the variation among the replicates and the poor detection of gCYTb5- III on western blots. The protein level of the flavohemoglobin also increased in response to the three stressors at the low concentrations of stressors that were tested. Only the cellular localization of gCYTb5-I changed in response to nitrosative stress, where it moved from the nucleolus to the nucleus and cytoplasm. This response was extremely sensitive and occurred at the lower doses of the three stressors, suggesting that gCYTb5-I may be involved in a nucleolar- based stress response.

Giardia intestinalis is an anaerobic protozoan that lacks common eukaryotic heme-dependent respiratory complexes and does not encode any proteins involved in heme biosynthesis. Nevertheless, the parasite encodes several hemeproteins, including three members of the Type II cytochrome b5 sub-group of electron transport proteins found in anaerobic protist and amitochondriate organisms. Unlike the more well-characterized cytochrome b5s of animals, no function has been ascribed to any of the Type II proteins. To explore the functions of these Giardia cytochromes (gCYTB5s), I used bioinformatics, immunofluorescence microscopy (IFM) and co-immunoprecipitation assays. The protein-protein interaction in silico prediction tool, STRING, failed to identify relevant interacting partners for any of the Type II cytochromes b5 from Giardia or other organisms. Differential cellular localization of the gCYTB5s was detected by IFM: gCYTB5-I in the perinuclear space; gCYTB5-II in the cytoplasm with a staining pattern similar to peripheral vacuole-associated protein; and gCYTB5-III in the nucleus. Co-immunoprecipitation with the gCYTB5s as bait identified potential interacting proteins for each isotype. The most promising candidate is the uncharacterized protein GL50803_9861, which was identified in the immunoprecipitate of both gCYTB5-I and II, and which co-localizes with both. Structural analysis of GL50803_9861 using Swiss Model, Phyre2, I-TASSER and RaptorX predicts the presence of a nucleotide-binding domain, which is consistent with a potential redox role involving nicotinamide or flavin-containing cofactors. Finally, the protein GL50803_7204 which contains a RNA/DNA binding domain was identified a potential partner of gCYTB5-III. These findings represent the first steps in the discovery of the roles played by these proteins in Giardia.

Author Keywords: Cytochrome b5, Giardia intestinalis, Heme, Interactome, Protein structure prediction

To study the Giardia intestinalis cell cycle, counterflow centrifugal elutriation (CCE) was used to separate an asynchronous trophozoite culture into fractions enriched for cells at the different stages of the cell cycle. For my first objective, I characterized the appearance of a third peak (Peak iii) in our flow cytometry analysis of the CCE fractions that initially suggested the presence of 16N cells that are either cysts or the result of endoreplication of Giardia trophozoites. I determined that this third peak consists of doublets of the 8N trophozoites at the G2 stage of the cell cycle that were not removed effectively by gating parameters used in the analysis of the flow cytometry data. In the second objective, I tested the use of a spike with RNA from the GS isolate of Giardia as an external normalizer in RT-qPCR on RNA from CCE fractions and encystation cultures of Giardia from the WB isolate. My results showed that the GS RNA spike is as effective as the use of previously characterized internal normalizer genes for these studies. For the third objective, I prepared two sets of elutriation samples for RNA seq analysis to determine the transcriptome of the Giardia trophozoite cell cycle. I confirmed the results of the cell cycle specific expression of several genes we had previously tested by RT-qPCR. Furthermore, our RNA-seq identified many genes in common with those identified from a microarray analysis of the Giardia cell cycle conducted by a collaborator. Finally, I observed an overall <4 fold change in differentially expressed genes during the G1/S and G2/M phase of the cell cycle. This is a modest change in gene expression compared to 10 - 30 fold changes for orthologous genes in mammalian cell cycles.

Author Keywords: Cell cycle, Counterflow Centrifugal Elutriation, Flow Cytometry, RNA-sequencing, RT-qPCR

Ustilago maydis is a biotrophic fungal plant pathogen that causes 'common smut of corn' disease. During infection, U. maydis develops a metabolic dependency on its host, relying on uptake of the carbon molecules provided within Zea mays tissues. The research presented indicated a requirement for metabolism of the pentose sugar D-xylose through functional investigation of a U. maydis xylitol dehydrogenase (uxm1), an enzyme involved in the bioconversion of D-xylose. This work is the first to outline the importance of pentose metabolism during biotrophic plant pathogenesis, as U. maydis haploid cells lacking this gene were impaired in their ability to cause disease and grow on medium containing only D-xylose. This thesis also explored the possibility that expression of this carbon-related gene is controlled by antisense RNAs (asRNAs), endogenous molecules with complementarity to mRNAs. Previous investigation of U. maydis asRNAs identified some that are exclusively expressed in the dormant teliospore, suggesting they have a functional role within this cell-type. A subset of these asRNAs at the uxm1 locus were investigated, with the purpose of identifying the mechanism(s) by which they influence U. maydis pathogenesis. This investigation involved the creation and functional analysis of a series of U. maydis deletion and expression strains. Together, these findings provided additional knowledge regarding the possible functions of U. maydis asRNAs, and their involvement in controlling important cellular processes, such as carbon metabolism and pathogenesis.

Author Keywords: antisense transcripts, fungal carbon metabolism, non-coding RNAs, pathogenesis, Ustilago maydis, xylitol dehydrogenase

Giardia lamblia is an intestinal parasite found globally in freshwater systems that is responsible for endemic outbreaks of infectious diarrhea. As a unicellular parasite that lacks mitochondria, a respiratory chain and lives in the anaerobic environment of its host's intestine, Giardia was assumed for decades to lack heme proteins. However, its genome encodes several putative heme proteins, including three with sequence similarity to the cytochrome b5 family, referred to as Giardia cytochromes b5 (gCYTb5). Recombinant expression of one of these genes (gCYTb5-I), results in a protein (17-kDa) that is isolated with noncovalently bound heme. Resonance Raman and UV-visible spectra of gCYTb5-I in oxidized and reduced states resemble those of microsomal cytochrome b5, while sequence alignment and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the heme iron. The reduction potential of gCYTb5-I measured by cyclic voltammetry is -165 mV vs the standard hydrogen electrode and is relatively low compared to those of other family members. The amino- and carboxy-terminal sequences that flank the central heme-binding core of the gCYTb5 are highly charged and do not occur in other family members. An 11-kDa core gCYTb5-I variant lacking these flanking sequences was also able to bind heme; however, we observe very poor expression of this truncated protein as compared to the full-length protein.

Author Keywords: b-type cytochrome, cytochrome b5, electron transfer protein, Giardia intestinalis, heme/heam protein, spectroelectrochemistry

The parasitic protist Giardia intestinalis lacks most heme proteins yet encodes a flavohemoglobin (gFlHb) that converts nitric oxide to nitrate and likely protects the cell from nitrosative stress. In this work an antibody raised against gFlHb was used to examine both changes in gFlHb expression levels and intracellular localization in Giardia in response to nitrosative stress. Giardia trophozoites exposed to stressors which either directly release nitric oxide (diethyltriamine NONOate, 1 mM) or are sources of other reactive nitrogen intermediates (sodium nitrite 20 mM or S-nitrosoglutathione, 1 or 5 mM) exhibited a 2 to 9-fold increase of gFlHb after 24 hours. Increased expression levels of gFlHb were detectable by 8 hours in S-nitrosoglutathione and diethyltriamine-NONOate-treated trophozoites, and by 12 hours after sodium nitrite exposure; these differences were likely due to differences in the rates of release of RNS from these compounds. In addition to a band of the expected size for gFlHb (52 kDa), western blots detected a second, higher molecular weight band (72 kDa) with comparable or higher intensity upon treatment with these RNS donors, which is consistent with sumoylation of gFlHb. Immunofluorescence microscopy of Giardia trophozoites detected gFlHb diffused throughout the cytoplasm and more punctuated staining along the cell membrane and between the nuclei. The punctuated staining may be due to the association of gFlHb with either peripheral vacuoles or basal bodies.

Author Keywords: Flavohemoglobin, Giardia intestinalis, Nitrosative stress

The amitochondrial protozoan, Giardia intestinalis, encodes four members of the cytochrome b5 (CYTB5) family of heme proteins of unknown function. While homology models can predict the likely fold of these proteins, supporting experimental evidence is lacking. The small size of the cytochromes (~16 kDa) makes them attractive targets for structural analysis by Electron Paramagnetic Resonance spectroscopy (EPR) and Nuclear Magnetic Resonance spectroscopy (NMR). EPR measurements are particularly useful in defining the geometry of the coordination environment of the heme iron; such measurements indicated that the planar imidazole rings of the invariant histidine axial ligands are nearly perpendicular to each other, rather than in the coplanar orientation observed within mammalian CYTB5s. This may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia cytochromes b5 (gCYTB5s). Following optimization of sample and instrument conditions for NMR experiments, a comparison of the 1D 1H-NMR spectra of gCYTB5 isotype I to those of three of its heme-pocket mutants (Tyr51→Phe, Tyr61→F, and Cys84→Ala) were used to tentatively assign the heme methyl and vinyl protons. Mutant Tyr61→F had the greatest effect on the wild-type spectrum due to maximum through-space contacts with the heme macrocycle and its proximity to the His63 axial ligand. These experiments are a prelude to further NMR experiments that can lead to solving the complete structures of these proteins.

Author Keywords: cytochrome b5, heme b, mutant protein, paramagnetic iron, resonant spectroscopy, sequence homology

Cytokinins (CKs) are a family of plant phytohormones responsible for many areas of plant growth and development. There are four free base types of CKs found in higher plants, trans-zeatin (tZ), N6-(∆2-isopentenyl)adenine (iP), cis-Zeatin (cZ) and dihydrozeatin (DZ). CK biosynthesis is regulated by adenosine phosphate-isopentenyltransferase (IPT), which is encoded by a multi-gene family in many plant species. There are two types of IPT pathways responsible for CK production, the tRNA pathway and the AMP (ATP/ADP) pathway. The tRNA pathway putatively produces cZ and the latter predominantly produces iP type nucleotides. CKs have long been studied for their role in stress tolerance, signal transduction, and involvement in many areas of plant growth and development. This study focuses on the role of CKs and CK biosynthesis by IPT during kernel development and comparisons of its regulation in high and low yielding barley and oat lines. The sequence of a putative IPT encoding gene in barley and oat was identified by a blast search of other known IPT gene fragments in closely related species. Quantitative Real time PCR results based on primers designed for the putative barley and oat IPT gene revealed changes in expression of IPT during different stages of kernel development, but no significance difference was associated with yield. Correlation of IPT gene expression in barley with cZ CK profiles measured by HPLC-MS/MS, confirms a putative IPT gene is a tRNA- IPT. HPLC-MS/MS results reveal some CK types, such as benzyladenine, are more predominant in higher yielding lines. This suggests different types of CKs play a role in yield production. Future studies on more IPT genes in the barley and oat IPT gene family will outline a more clear representation of the role of IPT in barley kernel development.

Author Keywords: Benzyladenine, Cereal grain, Cytokinin, Isopentenyl Transferase, Mass Spectrometry, Real Time PCR

The aim of this work was to identify the DNA sequences recognized by the Giardia TBP (gTBP) in vivo by using a chromatin immunoprecipitation assay (ChIP). Since a specific antibody for the protein of interest is required for this assay, a company was contracted to produce and purify a custom polyclonal antibody from the immunization of rabbits. Recombinant GST-gTBP was produced at a suitable yield and purity and used as the immunogen. The antibody was then tested for reactivity to the native protein in our laboratory. By Western blot analysis, it was possible to observe the enrichment of the gTBP within the nuclear fraction compared to a cytoplasmic fraction extracted from Giardia cells. However, the antibody could not be successfully used in an immunoprecipitation assay - suggesting that the antibody is unable to bind to the native structure of gTBP. Therefore, the focus of this work was changed to analyse gTBP via multiple sequence alignments, homology modelling and BLAST to identify any unique regions that may contribute to its unusual binding characteristics. These techniques were also used to identify specific regions of gTBP that may be used to generate synthetic peptides as immunogens for future antibody production.

Author Keywords: ChIP, Giardia intestinalis, Homology modelling, Immunoprecipitation, TATA-binding protein, Western Blotting

The flavohemoglobin in Giardia intestinalis (gFlHb) is the only known protozoan member of a protein class typically associated with detoxifying nitric oxide (by oxidation to nitrate) in bacteria and yeast. Mutants of the B10 tyrosine (Y30F) and E11 leucine (L58A), conserved residues thought to influence ligand binding, were expressed and studied using Resonance Raman (RR) spectroscopy. In the wild type protein, RR conducted using a carbon monoxide probe detects two distinct Fe-CO stretches associated with two different active site configurations. In the open configuration, CO does not interact with any polar side chains, while in the closed configuration, CO strongly interacts with one or more distal residues. Analysis of the Y30F mutant provided direct evidence of this tyrosine's role in ligand stabilization, as it had only a single Fe-CO stretching mode. This stretching mode was higher in energy than the open conformer of the wild type, indicating a residual hydrogen bonding interaction, likely provided by the E7 glutamine (Q54). In contrast the L58A mutant had no effect on the configurational nature of the enzyme. This was unexpected, as the side chain of L58 sits atop the heme and is thought to regulate the access of distal residues to the heme-bound ligand. The similar spectroscopic properties of wild type and L58A suggest that any such regulation would involve rapid conformational dynamics within the heme pocket.

Author Keywords: B10 Tyrosine, Catalytic Globin, E11 Leucine, Flavohemoglobin, gFlHb, Giardia intestinalis