Biochemistry

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

Methylobacterium spp., a dominant and functionally conserved group of plant-associated bacteria, have long been recognized for their roles in promoting host growth, stress tolerance, and phytohormone modulation. This body of work collectively repositions Methylobacterium not only as a plant growth-promoting genus but also as a promising agent of microbiome-mediated crop protection. Across several investigations, the ecological, biochemical, and functional attributes that underpin this potential were examined, with specific focus on hormone production, compatibility with agrochemical inputs, and antifungal activity.

A comprehensive inventory of 46 Methylobacterium strains revealed widespread production of cytokinins – including highly active forms such as trans-zeatin – and variable capacities to synthesize indole-3-acetic acid. Cytokinin output increased under carbon-limiting conditions, highlighting the genus's adaptive hormonal response. Parallel investigations demonstrated that commercial glyphosate-based herbicide formulations significantly inhibited the growth of most Methylobacterium strains, whereas pure glyphosate alone showed negligible toxicity. Key findings of experiments indicate that non-active formulation components participate in the disruption of beneficial bacteria by facilitating higher intracellular glyphosate concentrations and subsequent toxic effects. This introduces a novel link between agrichemical formulation practices and the selective disruption of keystone microbial taxa.

Contrastingly, fungicide compatibility testing showed that Methylobacterium strains tolerate key fungicides such as azoxystrobin, fludioxonil, and metalaxyl-M, supporting their inclusion in integrated pest management frameworks. Subsequent functional antagonism assays further revealed that specific Methylobacterium isolates inhibit phytopathogenic Fusarium species in vitro and in planta. Notably, M. organophilum enhanced soybean seedling vigor and reduced disease severity when co-inoculated with F. graminearum by preserving the integrity of the seed coat, demonstrating protective activity with unique mechanics.

Finally, differential hormone profiling at the pathogen-antagonist interface revealed that biocontrol-effective Methylobacterium strains not only produce higher levels of auxin and salicylic acid but also induces jasmonic acid production – likely derived from Fusarium – suggesting complex cross-signalling and interference with fungal development and sensing pathways. Together, these findings advance our understanding of Methylobacterium as a keystone genus in the phytobiome, capable of contributing to both plant vigor and pathogen suppression and reinforce its relevance in the design of next-generation biocontrol strategies.

Author Keywords: agrochemical interactions, biological control, Fusarium antagonism, Methylobacterium, phytobiome, phytohormone signalling

Fungi are a diverse group of organisms that play crucial roles in various ecological processes and have immense economic importance. Understanding the intricate mechanisms underlying fungal growth and development is fundamental to harnessing their potential and exploring their applications in different fields. Signalling molecules, such as hormones, have been identified as key regulators in fungal physiology, orchestrating intricate processes and modulating biological responses to the environment. Phytohormones, commonly associated with plants, have been proposed as potential myco-hormones due to their production in a wide variety of fungi. Metabolomic analyses were performed via LC-MS/MS to investigate the role of phytohormones, specifically cytokinins (CKs) and indole-3-acetic acid (IAA), along with lipids and energetic metabolites such as organic acids during the growth and development of the model fungus Sordaria macrospora. The results revealed a clear switch between CK ribosides and CK free bases during the ascosporegenesis stage, with increased levels of cZ and iP and decreased levels of iPR and cZR. A similar pattern was observed in the mutant strain smgpi1 but demonstrating higher levels of CK free bases and increased fruiting body formation compared to the wild type. These findings provide insights into the regulation of phytohormones especially during fungal fruiting body development. In terms of IAA, the levels increased during the transition to sexual development in all strains, with the per5 mutant, unable to produce lipids via the cytosol, demonstrating a higher concentration than the wild type. The interplay between energetic metabolites and IAA suggests a potential role in the transition to sexual development. Additionally, the dose-dependent effects of exogenous CK application were investigated, showing the potential of low concentrations, from 1 to 10 μM, of CKs in promoting biomass accumulation or sexual development. Furthermore, gene editing in S. macrospora was proposed as a future direction to explore the functions of CKs and other metabolites during fungal development. Overall, this research contributes to our understanding of phytohormone-mediated processes in fungi and opens avenues for future investigations in fungal biology.

Author Keywords: fungi, LC-MS, metabolomics, myco-hormone, phytohormone, sexual development

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of neurodegenerative diseases that affect all ages, primarily children. Batten disease is caused by mutations in 1 of the 13 ceroid lipofuscinosis neuronal (CLN) genes (CLN1-CLN8, CLN10-CLN14), each of which causes an NCL subtype when mutated. One of the NCL subtypes, CLN5 disease, is caused by mutations in the CLN5 gene. CLN5 is a soluble lysosomal protein that localizes to the endoplasmic reticulum (ER), the Golgi complex, the cytoplasm, and extracellularly. CLN5 has four putative molecular functions, including as a ceramide synthase, glycoside hydrolase, depalmitoylase, and bis(monoacylglycerol)phosphate synthase. CLN5 plays various roles within the cell, such as lipid metabolism, autophagy, and proteasome degradation. However, the function and the exact pathway in which CLN5 is involved are unclear. In addition, CLN5 is a secreted protein that, as shown via bioinformatics analysis, contains a signal peptide sequence. Furthermore, there are currently 70 CLN5 disease-causing mutations reported in the NCL mutation database. 12 CLN5 disease-causing mutations have been studied thus far in terms of their cellular impact, as well as the release of CLN5 to a certain extent. However, there is a lack of research into the functionality of the signal peptide in CLN5 and an in-depth analysis of the molecular impact of mutations in CLN5 disease. Consequently, this Ph.D. thesis focused on using comparative transcriptomics to reveal biological pathways affected by cln5-deficiency, revealing mechanisms that regulate the secretion of Cln5 and CtsD, and using Dictyostelium to gain insights into the molecular effects of mutations in CLN5 disease. Comparative transcriptomics reveal many differentially expressed genes that are linked to phenotypes observed in cln5-deficient cells and identified pathways affected in other CLN5 disease models, such as autophagy. Furthermore, novel findings, like affected expression of lysosomal enzymes and pathways, including secretion, are identified within the comparative transcriptomics analysis. Subsequently, this research also shows the secretory role of the signal peptide in Cln5 and CtsD. Finally, this Ph.D. thesis revealed that mutations in CLN5 disease affect the lysosomal biology and secretion of Cln5 and other lysosomal enzymes.

Author Keywords: Batten disease, CLN5, Dictyostelium discoideum, Enzymes, Lysosome, Secretion

Giardia intestinalis is a parasitic protozoan that possesses a flavohemoglobin (gFlHb), an enzyme that plays a role in the detoxification of reactive nitrogen species (RNS) and reactive oxygen species (ROS) via its nitric oxide dioxygenase (NOD) activity as well as its NADH-oxidase activity. This enzyme is a potential target for imidazole-based antigiardial drugs that act as ligands of the iron within its heme cofactor. In this work, two rapid and relatively inexpensive assays, the colorimetric Griess assay and a fluorescence assay, were adapted, optimized, and implemented to screen for flavohemoglobin inhibitors in parallel studies that compared the response of gFlHb to that of Hmp (Escherichia coli flavohemoglobin) when a group of six different imidazole-based compounds was tested. These assays displayed isotype selectivity, showing how the different drugs elicited different responses from the two enzymes. Comparative results for gFlHb and Hmp revealed that bulkier compounds elicited higher inhibition of Hmp, while smaller compounds resulted in better inhibition of gFlHb, which might be explained by the presence of different amino acid residues in the active sites of the enzymes, with two large amino acid sequence inserts being a unique feature of gFlHb, thus blocking the active site from being reached and blocked by larger compounds.

Author Keywords: 2.3-diaminonaphthalene, Flavohemoglobin, Giardia intestinalis, Griess Assay, imidazole-based drugs, nitric oxide detoxification

Flavohemoglobins are enzymes primarily implicated in nitrosative stress due to their high nitric oxide (NO) dioxygenase activity and transcriptional upregulation in response to NO. Giardia intestinalis assemblages A, B, and E possess flavohemoglobins (gFlHb) that may function beyond their NO dioxygenase activity, potentially contributing to oxidative stress regulation, as transcriptional profiling revealed that peroxide also induces gFlHb expression. This study investigates gFlHb's NADH oxidase activity in the absence of NO, structural interactions with lipids, and response to reactive oxygen species. Minor differences in NADH oxidase activity among assemblages were observed, and their susceptibilities to inhibition were assessed to evaluate gFlHb as a potential therapeutic target against Giardia infection. Under aerobic conditions, we observed that gFlHb generates hydrogen peroxide, a surprising finding suggesting a self-regulating feedback mechanism involving reactive oxygen species and heme degradation. These findings provide new insight into the role of flavohemoglobins in microaerotolerant parasites like Giardia.

Author Keywords: flavohemoglobin, Giardia intestinalis, heme, hydrogen peroxide, NADH, oxidative stress

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 accelerated recovery of base-poor soils from the legacy effects of acidic deposition may be possible by applying industrial wood ash as a soil amendment. Wood ash may be an effective soil amendment due to its high alkalinity and concentrations of several essential nutrients, such as calcium, magnesium, potassium, and phosphorus, that are retained after the volatilization of the parent material. However, wood ash can also contain trace amounts of metals that could be released into the soil and soil solution. The short-term (<3 years) biogeochemical response of soils, microbial communities, and sugar maple (Acer saccharum Marsh.) trees were assessed following wood ash application at Porridge Lake, Ontario. The study design consisted of five blocks containing three treatment plots each (2.5, 5.0, 7.5 Mg ha-1) and a control. Soil solution pH, base cation, and trace metal concentrations were monitored for three years, using tension lysimeters at depths of 30 and 60 cm and zero-tension lysimeters for forest floor percolate within each plot. In the last year of the trial, soil, foliage, and fine root samples were collected and analyzed for trace elements. Also, soil samples were analyzed for the abundance of 16S and ITS DNA through metabarcoding to ascertain the microbial response to wood ash. Significant changes in soil solution pH were measured within the forest floor horizon in the first year of the trial. Significant increases in calcium (Ca), magnesium (Mg) and calcium/aluminum (Ca/Al) ratios were also observed in the second year of the trial, along with decreases in dissolved organic carbon (DOC), sulphate (SO4) and nitrate (NO3) in the LFH horizon. By the third year of the trial, significant increases in soil solution pH and potassium (K) concentrations and decreases in Al were observed to a depth of 30 cm. Changes in trace metal concentrations in soil water were notably variable, with concentrations of chromium, copper, lead, nickel, and selenium remaining unresponsive, whereas concentrations of cadmium, manganese and zinc decreased by the third year. The metalloid arsenic showed a significant increase in the third year of the trial but remained below regulatory guidelines, similar to all other trace metals. Soil measurements conducted in the third year of the trial showed positive pH responses in the FH horizon and increases in Ca and Mg in the Ah and Bm soil horizons, but foliar base cation and metal concentrations were unchanged. Diversity analysis on the soil prokaryotic and eukaryotic groups indicated increased bacterial alpha diversity in the FH horizon and bacterial dominance in the litter horizon. Analysis of relative abundance at the phylum level for prokaryotes and at the order for eukaryotes did not indicate any compositional shifts due to the wood ash treatments. Changes in the length and diameter of sugar maple and mycorrhizal fine root may point to pH shock being an issue at higher ash doses. The results from this study indicate that wood ash has a strong ameliorative effect on soil properties and does not pose a risk to soil communities. 

Author Keywords: DNA, mycorrhizae, soil acidication, soil amendments, soil solution, sugar maple

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

Fungi are a diverse group of organisms that play crucial roles in various ecological processes and have immense economic importance. Understanding the intricate mechanisms underlying fungal growth and development is fundamental to harnessing their potential and exploring their applications in different fields. Signalling molecules, such as hormones, have been identified as key regulators in fungal physiology, orchestrating intricate processes and modulating biological responses to the environment. Phytohormones, commonly associated with plants, have been proposed as potential myco-hormones due to their production in a wide variety of fungi. Metabolomic analyses were performed via LC-MS/MS to investigate the role of phytohormones, specifically cytokinins (CKs) and indole-3-acetic acid (IAA), along with lipids and energetic metabolites such as organic acids during the growth and development of the model fungus Sordaria macrospora. The results revealed a clear switch between CK ribosides and CK free bases during the ascosporegenesis stage, with increased levels of cZ and iP and decreased levels of iPR and cZR. A similar pattern was observed in the mutant strain smgpi1 but demonstrating higher levels of CK free bases and increased fruiting body formation compared to the wild type. These findings provide insights into the regulation of phytohormones especially during fungal fruiting body development. In terms of IAA, the levels increased during the transition to sexual development in all strains, with the per5 mutant, unable to produce lipids via the cytosol, demonstrating a higher concentration than the wild type. The interplay between energetic metabolites and IAA suggests a potential role in the transition to sexual development. Additionally, the dose-dependent effects of exogenous CK application were investigated, showing the potential of low concentrations, from 1 to 10 μM, of CKs in promoting biomass accumulation or sexual development. Furthermore, gene editing in S. macrospora was proposed as a future direction to explore the functions of CKs and other metabolites during fungal development. Overall, this research contributes to our understanding of phytohormone-mediated processes in fungi and opens avenues for future investigations in fungal biology.

Author Keywords: fungi, LC-MS, metabolomics, myco-hormone, phytohormone, sexual development