Browsing by Author "Suh, Dae-Yeon"

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Open Access
Anti-candida and Anti-virulence Activity of Essential Oils and Their Components
(Faculty of Graduate Studies and Research, University of Regina, 2021-10) Shahina, Zinnat; Dahms, Tanya; Suh, Dae-Yeon; Babu, Mohan; Cameron, Andrew; Lee, Samuel A.
Candida albicans is one of the most common fungi associated with lifethreatening infections. Treatment of the associated infections is often ineffective in the light of resistance, and so there is an urgent need to discover novel antifungals. Rather than killing the fungal cells, which requires quite high specificity and can lead to the emergence of further resistance, inhibiting growth and virulence factors in fungal cells represents a good alternative for the development of new antifungal drugs. Recently, there has been a resurged interest in essential oils and their active components, in relation to their pharmacological properties. The primary objective of this research was to evaluate the antifungal activity of cinnamon bark and rosemary essential oils, along with its major components cinnamadehyde, and 1,8-cineole, α-pinene, respectively, and two other common essential oil components, namely eugenol and citral, against C. albicans. Despite many reports on the antimicrobial activities of essential oils, the results have been diverse and the evaluation methods inconsistent. At higher essential oil concentrations, reactive oxygen species were generated, impacting a wide range of processes including cell membrane depolarization, vacuolar segregation, mitochondrial dysfunction, cell-cycle checkpoint deficiency, and mitotic catastrophe, which resulted in C. albicans death. At lower fractional MICs, essential oils had a ROS-independent response that inhibited mycelial growth and biofilm formation, which may be attributable to defects at the cell membrane. However, microtubule inhibition also plays a role in limiting hyphal growth. I explored the microtubule defects and how they relate to Kar3p, a member of the kinesin-14 family shown to be linked to microtubule stability. Interestingly, both tubulin and Kar3 protein was delocalized with essential oil components exposure at levels for which endogenous ROS levels were normal. I further demonstrate that the level of Kar3 is associated with resistance and susceptibility of C. albicans to essential oil components in both liquid and on solid growth media. All mutant strains grown on hyphae-inducing media in the presence of essential oil components exhibited an enhanced tendency to form pseudohyphal cells, a common phenotype for the homozygous and heterozygous deletion strains. Towards these ends, microtubule defects were linked with theoretical binding between essential oil components and α-tubulin and Kar3p adjacent to cofactor binding sites, consistent with experimentally observed hyphal defects and biofilm inhibition. This study for the first time uncovers a new mode of essential oilmediated microtubule defects, which does not follow the known mechanisms of conventional microtubule inhibition. The essential oil components appear to impact C. albicans eventually giving rise to psuedohyphal formation, microtubule loss, hyphal and biofilm reduction. Since those essential oil components impact multiple Candida targets, they should be less susceptible to resistance. Further, many components showed antivirulence, and may represent an effective approach for inhibiting Candida, especially in the context of physically preventing Candida from entering its host through catheters and prosthetics.
Open Access
Characterizaion and Chemical Rescure of the Phenotype of ors-3, a Knockout mutant line of PpORS in Physcomitrella patens
(Faculty of Graduate Studies and Research, University of Regina, 2019-04) Aslam, Misbah; Suh, Dae-Yeon; Babu, Mohan; Ashton, Neil; Weger, Harold
2ʹ-Oxoalkylresorcinol synthase from Physcomitrella patens (PpORS) is basal to all plant type III polyketide synthases in phylogenetic trees, and may resemble closely their most recent common ancestor. PpORS knockouts were previously generated and partially characterized (Li et al., Planta, 2018, 247: 527–541). This study aimed to investigate further the in planta functions and evolutionary roles of PpORS. Ors-3 (an ors knockout line) was first subjected to dehydration stress. The ability of ors-3 to recover after dehydration is significantly compromised in contrast to the control strain. Ors-3 also loses water faster than the control strain. These results together with the previous data suggested that ors mutants possess a defective cuticle (Li et al., 2018). In ors-3, the expression of two putative paralogs of PpORS was also examined to reveal that their expression levels did not appear to be up-regulated in ors-3 as compared to those in the control. Chemical rescue of ors-3 has been achieved. Thus, the ability of ors-3 to survive dehydration is restored in a dose dependent manner by in vitro PpORS products, purified oxoalkylresorcinol, and also by long chain alkylresorcinol analogs. Exogenous 14Clabelled in vitro PpORS products are incorporated as an insoluble biopolymer, and most of the radoioactivity was recovered after acid hydrolysis. Taken together, these data indicate that PpORS-produced 2ʹ-oxoalkylresorcinols are constituents of the moss cuticular biopolymer that confer resistance to dehydration, and imply that an ancestral ORS in early land plants may have contributed to their successful colonization of the land.
Open Access
Cyclization modes in type III polyketide synthases and the synthetic compounds used as probes for mechanistic studies.
(Faculty of Graduate Studies and Research, University of Regina, 2012-08-20) Posehn, Sarah Elizabeth; Suh, Dae-Yeon; Wee, Andrew; Sterenberg, Brian; Young, Stephanie
Type III polyketide synthases (PKS) catalyze different cyclization reactions of linear polyketide intermediates to produce polyketides with distinct ring structures. The mechanisms of pyrone and resorcinol ring formation in type III PKSs remain unknown, despite many years of research on this family of enzymes. Compounds, thought to be intermediates in these enzyme reactions, have been synthesized and used to probe the enzyme mechanisms. To study the mechanism of resorcinol ring formation catalyzed by STS and ArsB, triketo acids and 9-phenyl-3,5,7-trioxo-8-nonenoic and 3,5,7-trioxoeicosanoic (16a and 16b) and their structural analogs were synthesized. STS failed to produce any cyclized products from the synthetic compounds. ArsB also failed to convert 3,5,7-trioxoeicosanoic acid 16b to 5-tridecylresorcinol 14b. Instead, ArsB facilitated the production of 6-tridecylresorcylic acid 11b from the synthetic 16b and hindered the decarboxylation of 11b to 14b. These effects were also observed with ArsC, another acyl-CoA utilizing PKS that does not catalyze the resorcinol ring formation. Furthermore, the triketo acid 16b and its analogs inhibited the ArsB activity. These results indicate that neither 16b nor 11b is an intermediate and that aldol cyclization of the linear tetraketide thioester intermediate (15) precedes the hydrolysis of 15 in the ArsB catalyzed resorcinol ring formation. 3,5-Dioxooctanoic acid 32a was also synthesized in order to probe the lactonization mechanism in type III PKSs such as PpASCL and AthPKSA. It has been suggested that diketo acids are synthesized by these enzymes, released, and then lactonized in solution. The results of this study show that even prolonged incubation of 32a in buffer failed to result in any pyrone formation. Also, the enzymes were unable to convert the 32a to 4-hydroxy-6-tridecyl- 2-pyrone 33a, which led to the conclusion that pyrone formation is most likely an enzymatic process, occurring via direct lactonization of a CoA thioester.
Open Access
Defective Spore Development and Differentially Expressed Genes In The Sporophytes of mkn4-2-1, A Triple Deletion Mutant of Physcomitrella patens
(Faculty of Graduate Studies and Research, University of Regina, 2020-05) Akther, Farhana; Suh, Dae-Yeon; Ashton, Neil; Dahms, Tanya; Weger, Harold
KNOTTED1-like homeobox (KNOX) genes encode homeodomain transcription factors involved in maintaining the shoot apical meristem of flowering plants. They are widely distributed in land plants where they perform diverse roles. A triple knockout of KNOX genes, mkn4-2-1, in the moss, Physcomitrella patens, produced malformed sporophytes and defective spores with under-developed perine (Singer and Ashton, 2007, Plant Cell Rep, 26: 2039). I hypothesized that moss KNOX homologues (MKN genes) could be the executive regulators of spore wall formation in Physcomitrella, and that the biosynthesis and regulatory networks of spore/pollen grain wall formation might be conserved in plants. The mkn4-2-1 spores shed their walls, and underwent augmented osmolysis. Although defective, the exine in mkn4-2-1 spores is more resistant to alkaline hydrolysis than that of control spores. Mkn4-2-1 produced malformed tetrads and fewer spores per sporophyte. The spores were heterogeneous in size and shape, suggesting defective meiosis of mkn4-2-1 sporocytes. Interestingly, the polymeric and, possibly, lignin-like pigments in the setae of the mutant were alkaline sensitive, suggesting their defective biosynthesis. Based on phenotypic observations and available transcriptomic data, a total of 111 Physcomitrella genes were selected as putative target genes of MKN1, 2 and/or 4, and their differential expression levels in mkn4-2-1 and control sporophytes were investigated by semi-quantitative RT-PCR. Physcomitrella homologues of Arabidopsis genes responsible for sporopollenin deposition (NEF1, NPU), tapetum development (EMS1, TPD1, MS1), and spore wall formation (CUSL) were down-regulated in mkn4-2-1, implicating them in defective spore wall formation. RT-PCR revealed reduced expression of meiosis-specific genes DMC1, ASY1 and SPO11 in mkn4-2-1, providing a ii possible explanation for abnormal tetrad formation, heterogeneous spores and fewer spores per sporophyte. Reduced expression was also found in genes involved in the metabolism of lignin-like materials (CCR, LACL1, LACL2). This study uncovered novel phenotypic differences in spore walls, tetrad formation and sporophytic pigments in the mutant, and identified several differentially expressed genes that may be regulated by MKN1, 2 and/or 4. It also provides insights into the functional evolution of KNOX genes in plants and the regulatory roles of MKN genes in sporophytic ontogeny, sporogenesis and the formation of the spore wall in a moss.
Open Access
An Efficient Lentiviral-based Proteomics Approach Identifies Mitochondrial Complexes with Neurodegenerative Function
(Faculty of Graduate Studies and Research, University of Regina, 2015-04) Kassir, Sandy; Babu, Mohan; Suh, Dae-Yeon; Dahms, Tanya; Buttigieg, Josef
Perturbations in proteins involved in the mitochondrial processes have emerged as a causative factor in a wide range of life-threatening human diseases, such as neurodegenerative disorders. In fact, mitochondrial dysfunctions are argued to be the first step in triggering the onset of other deleterious events that collectively act causally in the pathogenesis of diseases. Accordingly, dysfunctions in mitochondrial proteins influence the proper functioning of basic processes, such as energy metabolism and reactive species production. Indeed, biochemical and genetic evidence has provided molecular insights into the role of mitochondrial proteins and complexes in isolation, yet our understanding of how mitochondrial proteins cause and/or contribute to the diverse array of human diseases, including neurodegeneration remains unclear. Like any other biological system, mitochondria are linked together by extensive networks of physical (protein-protein) interactions; therefore a detailed understanding of the systems properties is required to unravel their role in neurodegenerative diseases. To address this, I have developed and optimized an effective procedure to identify the physically interacting proteins for generating a mitochondrial protein-protein interaction network for those involved in various neurodegenerative diseases.
Open Access
Evaluation of Antibiotic Production and its Regulation in Pantoea agglomerans Tx10
(Faculty of Graduate Studies and Research, University of Regina, 2015-08) Robinson, Lucas; Stavrinides, John; Yost, Christopher; Suh, Dae-Yeon
The prevalence of antimicrobial-resistant pathogens, such as methicillinresistant Staphylococcus aureus (MRSA), has prompted numerous efforts to identify new antimicrobials. One untapped source of antimicrobial products is the enterobacterial genus Pantoea. Although antibiotic discovery efforts in Pantoea have focused largely on agricultural applications, Pantoea remains an unexplored reservoir of antibiotics that may have utilization in a clinical setting. Using an agar overlay assay, Pantoea agglomerans Tx10 (Tx10)—an isolate from the sputum of a cystic fibrosis patient—was shown to inhibit the growth of several pathogens, including Gram-positive Staphylococcus and Streptococcus, and the Gram-negative Citrobacter, E. coli, Erwinia, and Salmonella. A genetic screen to identify the genes involved in the production of this antimicrobial natural product identified a predicted 6-gene biosynthetic cluster which, when disrupted, abolished antibiotic production. I called this compound, Pantoea Natural Product 2 (PNP-2). Subsequent assays with mutants deficient in PNP-2 production revealed that they were still able to inhibit Erwinia amylovora, suggesting the production of a second antibiotic, which I identified as the well-known pantocin A. A survey of resistance among the Stavrinides Pantoea collection revealed that the majority of isolates are susceptible to either pantocin A, or PNP-2, and only a minority were resistant to both. Analysis of the expression of PNP-2 revealed it was produced only on minimal media. Using the luciferase reporter gene, PNP-2 expression was shown to be linked to amino acid starvation, with the absence of certain amino acids enabling antibiotic production. Overall, the antibiotic production from this cystic fibrosis isolate, Tx10, shows the potential reservoir of antibiotics from the genus of Pantoea, as well as insight into the production and expression of the antibiotic in the environment.
Open Access
Identification and Characterization of Antibiotic Biosynthetic Gene Clusters in Pantoea
(Faculty of Graduate Studies and Research, University of Regina, 2021-08) Williams, Ashley; Stavrinides, John; Weger, Harold; Cameron, Andrew; Suh, Dae-Yeon; Ruzzini, Antonio
Antimicrobial resistance is a global health crisis for which new antibiotics are needed. Natural products (secondary metabolites) produced by microbes have been our primary source of antibiotics; however, the search has been predominantly limited to a few genera. One underexplored bacterial genus is Pantoea, which produces a variety of compounds with antimicrobial activity. Chapter one describes a general overview of secondary metabolites, antibiotic resistance, strategies to overcome resistance, and Pantoea. Chapter two describes the identification and characterization of the biosynthetic gene cluster for Pantoea Natural Product 3 (PNP-3), an antibiotic produced by P. agglomerans 3581r and SN01080r. The cluster contains putative enzymes (pnp3b, pnp3e–h), MFS transporters (pnp3a, pnp3c), and an ArsR family regulator (pnp3d). Transposon mutagenesis and heterologous expression of pnp3e–h showed that the four genes are sufficient for antibiotic production. Disruption by homologous recombination of the putative hydrolase pnp3b increased the PNP-3 zone of inhibition, suggesting Pnp3b may act as a resistance mechanism. PNP-3 activity was demonstrated to be dependent upon low nutrient levels and was broad-spectrum, inhibiting the growth of drug-resistant bacterial strains including Acinetobacter baumannii and Pseudomonas aeruginosa. Further, the PNP-3 gene cluster has a limited distribution across bacterial species, and genome mining tools were unable to identify the cluster, suggesting the metabolite may be novel. Chapter 3 describes the characterization of the two predicted MFS transporters, Pnp3a and Pnp3c, using genetic, phylogenetic, and bioinformatic approaches. Disruption via homologous recombination of the putative transporters in Pantoea suggests a role of Pnp3a in the export of the metabolite. This was supported by experiments where heterologous expression of Pnp3a in E. amylovora conferred resistance to PNP-3 and facilitated the export of the metabolite produced by Pnp3e–h. The role of Pnp3c, however, is unclear as it appears to be dispensable under certain conditions. Comparative genomic analyses identified pnp3a in additional Pantoea strains, several of which carry complete or nearly complete PNP-3 biosynthetic clusters. Two Pantoea strains with PNP-3 clusters (P. vagans C9-1, P. agglomerans SS03231r) were able to inhibit the growth of P. aeruginosa, suggesting the PNP-3 biosynthetic clusters are functional, and all strains carrying a pnp3a homolog, regardless of genomic context, were tolerant to PNP-3. These results suggest Pnp3a plays an essential role in PNP-3 export and resistance. Chapter four describes an antibiotic production survey of 116 Pantoea strains targeting 12 bacterial strains, including several human pathogens. Of the Pantoea strains tested, 59 were antibiotic producers. Among the antibiotic producers was P. agglomerans B025670, which was antagonistic toward Kosakonia, E. coli, Salmonella, Enterobacter, and Pseudocitrobacter species. To identify the antibiotic biosynthetic gene cluster responsible for antibiotic production in B025670, an integrated approach of comparative genomics, genome mining, and functional genetics was employed. The approach identified a 14-gene cluster in the genome of B025670, designated cluster 675. Disruption of the gene cluster via single-integration homologous recombination led to a loss of antibiotic production, confirming a role in the manufacturing of the antimicrobial product. Chapter 5 describes the general thesis conclusions and future directions. Overall, this research has sought to characterize antibiotic biosynthetic gene clusters in Pantoea and highlights the genus as a promising source of antibiotics for further exploration.
Open Access
Identifying Pantoea Genetic Factors Involved in Host Association by Integrating Comparative Genomics with a Dictyostelium discoideum Grazing Resistance Model
(Faculty of Graduate Studies and Research, University of Regina, 2018-10) Smith, Derek David Norman; Stavrinides, John; Cameron, Andrew; Chao, Tzu-Chaio; Suh, Dae-Yeon; Van Hamme, Jon
Pantoea is a genus of Gram-negative bacteria within the Enterobacterales. Much of what is known about Pantoea has been determined through studies focused on its phytopathogenicity and epiphytic associations. Limited research has been conducted on Pantoea to characterize its capacity for opportunism in humans, despite numerous reports of clinical infections. This thesis aims to identify genetic factors that could contribute to host association and thus, opportunistic infections by Pantoea. Chapter 1 provides a broad literature review of Pantoea, opportunism and virulence, model systems to assess virulence, and the field of genomics. Chapter 2 describes the adaptation of a model system using an amoebal predator, Dictyostelium discoideum, to screen bacteria for candidate virulence factors. Ten strains of Pantoea ananatis were screened for resistance to amoebal grazing, which was used as a potential indicator for virulence. A highthroughput genetic screen was developed and performed with grazing-resistant P. ananatis BRT175. This screen identified genes involved in quorum sensing, periplasmic protein redox maintenance, anaerobic metabolism regulation, and nucleotide biosynthesis. A novel hexose-based glycolipid biosurfactant, produced by the rhlA and rhlB genes in P. ananatis BRT175, was identified as being key for both swarming motility and grazing resistance. However, the rhlA and rhlB genes were present in both grazing-resistant and -susceptible strains. Chapter 3 addresses the differential regulation of rhlA in Pantoea. Variants within the putative promoter region of rhlA were identified in the 10 P. ananatis strains with an allele being correlated with grazing resistance. The promoters of rhlA from both P. ananatis BRT175 and P. stewartii DC283 were cloned into a lux reporter plasmid. The P. ananatis BRT175 rhlA promoter showed higher expression at 21°C compared to 30°C and 37°C. The P. stewartii DC283 rhlA promoter was less active, both in P. stewartii DC283 and P. ananatis BRT175, in hrp inducing medium than the native rhlA promoter from P. ananatis BRT175. Chapter 4 explores the genes associated with grazing resistance in the 10 P. ananatis strains using multiple comparative genomic techniques. This included kmer analysis with Neptune and gene presence/absence analyses with Roary and OrthoMCL to identify additional virulence factors to explain the spectrum of grazing resistance within P. ananatis. Multiple candidate genes were identified in subsets of resistant strains including formate dehydrogenase-N, which may increase fitness when dealing with phagocytes. P. ananatis virulence factors that differentiate it from P. stewartii were also determined. All 10 P. ananatis strains have the genes, yhcF and yhcA, which are part of putative fimbrial biogenesis cluster that may aid in host-specific adhesion. The pagC gene, a homologue of the Salmonella Resistance to Complement Killing (rck) virulence factor, was also present in P. ananatis but not in P. stewartii. SNP and indel analysis identified variation in the upstream region of the quorum sensing regulator eanI that was common to grazingsusceptible strains. This thesis identified multiple virulence factors in P. ananatis that have the potential to facilitate opportunistic infections of humans. This work also describes the genetic versatility of P. ananatis and highlights that its ability to colonize and persist in multiple host niches lies within both the conserved core and open pangenome.
Open Access
Plasticity of the heat shock response and development of thermotolerance during embryonic development of Lake Whitefish (Coregonus clupeaformis)
(Faculty of Graduate Studies and Research, University of Regina, 2015-09) Sessions, Katherine Jean; Manzon, Richard; Somers, Christopher; Weger, Harold; Suh, Dae-Yeon
Thermal discharge produced by industrial facilities has the potential to impact development of fish by increasing background temperature of the surrounding area. The overall objectives of this study were to: 1) Examine the interplay between how temperature and frequency of heat shock and post-heat shock recovery time can modulate the heat shock response to a subsequent more severe stressor; 2) determine if this interplay is the same at different embryonic stages; and 3) assess whether transient heat shocks confer protection to the embryo. These objectives were addressed by exposing embryos to repeated transient heat shocks and high-level heat shocks and then quantifying heat shock protein mRNA levels and whole animal responses such as, percent survival, time to hatch and morphometrics. Reverse transcription quantitative real-time PCR revealed that transient heat shocks increased or attenuated the heat shock response to severe heat shock depending on the post-transient recovery period. The most frequent transient heat shocks, every 3 days, had the lowest levels of hsp70 mRNA of all heat shock treatments. Embryonic stages had similar results, however, it seemed older embryos had a larger capacity to respond to heat shock as determined by greater increases in heat shock protein mRNA levels. Transient heat shock regimes were also able to confer protection to embryos exposed to a severe 4 h +18 °C high-level heat shock. In general, transient heat shock regimes seem to have some benefit to the embryo allowing for a quicker response to stress and therefore, greater tolerance.
Open Access
Role of Nudix-Hydrolase ASMTL in Modulating Mitochondrial Biogenesis of Cancer Cells
(Faculty of Graduate Studies and Research, University of Regina, 2021-05) Amin, Shahreen Tina; Babu, Mohan; Fitzpatrick, Dennis; Suh, Dae-Yeon; Weger, Harold; Kumar, Ashok
Cancer cells have enhanced DNA biosynthesis, rendering them susceptible to nucleotide modifications that enter the cellular nucleotide pool during repair or DNA degradation. These modified nucleotides can then be incorporated into newly synthesized DNA, resulting in random mutations or, when overwhelming into DNA damage, culminating into apoptosis, which is the desired effect of anti-cancer chemotherapy. Human cells contain nucleotide sanitation enzymes like ASMTL, which prevent incorporating the non-canonical nucleotides into newly synthesized DNA by removing them from the nucleotide pool, thereby relieving cancer cells from proliferative stress-induced mutations and apoptosis, representing an attractive target for anti-cancer chemotherapy. To identify ASMTL as a target for anti-cancer treatment, we investigated the ASMTL requirement for cancer survival in human cancer cell lines and patients, where ASMTL depletion decreased survival. This decrease in cell survival correlated with 14-3-3 interaction-dependent mitochondrial localization of ASMTL. Analyzing mitochondrial function suggests that ASMTL is imperative in the TP53 dependent BAX-BCL2 pathway, which is turned on by inefficient repair of mtDNA damage. Furthermore, after screening 2040 compounds, we identify small molecules TFBQ and TFHQ as ASMTL inhibitors that potently and selectively engage the ASMTL protein after occupying putative ASMTL MAF active site and impeding ASMTL-14-3-3 interaction. Finally, ASMTL is validated as an anti-cancer target in vivo where ASMTL knockout or inhibition triggers apoptosis and decreased metastasis in xenografts. This study collectively exemplifies the non-oncogene addiction concept for cancer treatment and validates ASMTL as phenotypically lethal to carcinomas.
Open Access
Role of β-galactofuranose and β-glucan in Aspergillus nidulans hyphal cell wall ultrastructure and physical properties
(Faculty of Graduate Studies and Research, University of Regina, 2012-07) Paul, Biplab Chandra; Dahms, Tanya; Suh, Dae-Yeon; Ashton, Neil; Stavrinides, John
The fungal cell wall is a first line of defence from the external environment or chemical treatments. Glucan, chitin and mannan are the main components of the Aspergillus nidulans hyphal cell wall. The sugar β-galactofuranose is a minor component of the cell wall and thought to be responsible for cross-linking of the other cell wall components, therefore responsible for maintaining cell wall structural integrity. We investigated the role of β-galactofuranose on the structure and physical properties of the hyphal cell wall. Based on its unique capacity to image live samples, atomic force microscopy was used to determine both the ultrastructure and physical properties of the hyphal cell wall. Five different knockout strains of Aspergillus nidulans (ugeAΔ, ugeBΔ, ugeAΔ,ugeBΔ and ugmAΔ, ugeAΔ,ugmAΔ and ugtAΔ) associated with β-galactofuranose synthesis were compared with the wildtype strain (AAE1). Atomic force microscopic imaging and force spectroscopy of the mutant and wild type strains suggest that a lack of galactofuranose reduces the integrity of cell wall components, where the surface subunits of ugeAΔ and ugmAΔ are two-four fold larger than that of the wildtype (AAE1) respectively. The ugeBΔ strain shows similar sized subunits as the AAE1 strain, in contrast with the double mutant (ugeAΔ,ugeBΔ) which exhibits a fibrous cell surface structure. The ugtAΔ mutant strain, able to synthesize β-Galf but unable to incorporate the sugar into the cell wall, showed a similar surface structure to the double mutant ugeAΔ,ugmAΔ, with the largest surface subunits. The structural changes of the cell wall surface are accompanied by a change in cell wall viscoelasticity, where the cell wall of the wild type strain is the most viscoelastic in comparison to that of mutant strains, and the lowest cell wall viscoelasticity can be attributed to the complete absence of β-Galf. Live and fixed cell walls of the ugmAΔ, ugtAΔ, ugeAΔ,ugeBΔ, and ugeAΔ,ugmAΔ strains had extremely low viscoelastic moduli, which we attributed to a limitation of the model used to accurately calculate viscoelasticity. Moreover, the impaired cell wall packing in mutant strains is consistent with greater surface hydrophilicity for mutant strains compared to wild type. We propose that the lack of galactofuranose disrupts the proper packing of cell wall components, giving rise to more disordered surface subunits and therefore greater deformability. Topographic images of glucanase- and laminarinase-treated wildtype strains suggest that glucan is at least one component of the cell surface subunits. Mutant strains which lack Galf were more susceptible to laminarinase treatment, which we attribute to deeper enzyme penetration into the more loosely packed cell walls.
Open Access
Studies on Syntheses of 1-Azaspirocycles, 2,6-Disubstituted 3-Piperidinols and cls-Decahydroquinolines
(Faculty of Graduate Studies and Research, University of Regina, 2015-07) Annadi, Krishna Reddy; Wee, Andrew G.; Suh, Dae-Yeon; East, Allan; Stavrinides, John; Gravel, Michel
Pyrrolidines and piperidines bearing a nitrogen-substituted quaternary stereocenter are commonly encountered structural motifs in many naturally occurring alkaloids, and therefore development of methods for their synthesis are important. The Rh(II)-catalyzed intramolecular tertiary C-H insertion reaction of 5-(-diazoacetoxy)piperidin-2-ones and 3-(-diazoacetoxy)pyrrolidines carrying C6 and C2 alkyl substituents, respectively, was investigated for the formation of bicyclic lactones bearing a nitrogen-substituted quaternary stereocenter. Both Rh(II)-carboxylate and Rh(II)-carboxamidate catalysts with varying electronic and steric properties were examined; however, these reactions usually resulted in the formation of complex mixture of products and the desired tertiary CH insertion products were not formed. The steric interactions encountered by the Rh(II)-carbenoid with the tertiary CH insertion site might have impeded the desired CH insertion reaction pathway. Alkylidenecarbene generation-1,5-CH insertion reaction of 5-(3-oxobutyl)pyrrolidin-2-ones and 6-(3-oxobutyl)piperidin-2-ones was next investigated for the formation of 7-methyl-2-azaspiro[4,4]non-6-ene-3-ones and 2-methyl-6-azaspiro[4,5]dec-1-ene-7-ones, respectively. The precursors 5-(3-oxobutyl)pyrrolidin-2-ones and 6-(3-oxobutyl)piperidin-2-ones required for this study were obtained from Wacker oxidation of 5-(but-2-enyl)pyrrolidin-2-ones and 6-(but-2-enyl)piperidin-2-ones, respectively with excellent regioselectivity (11:1). Further studies on Wacker oxidation have suggested that the lactam ring has a strong influence on the observed regioselectivity. Optimization studies for alkylidenecarbene generation-CH insertion reaction indicated that the efficiency of the CH insertion reaction is sensitive to the reaction temperature and the amount of lithio(trimethylsilyl)diazomethane employed. Under the optimized reaction conditions, 7-methyl-2-azaspiro[4,4]non-6-ene-3-ones and 2-methyl-6-azaspiro[4,5]dec-1-ene-7-ones were obtained in very good yields. The application of the developed method was successfully demonstrated in the total synthesis of both racemic- and ()-adalinine. The utility of a chiral non-racemic bicyclic lactam lactone (BLL) as a chiral building block was shown by developing approaches for the synthesis of two structural classes of alkaloids, the 2,6-disubstituted-3-piperidinols and cis-decahydroquinolines (cis-DHQs). The BLL was efficiently converted to (5S,6S)-5-hydroxymethyl-6-hydroxypiperidin-2-one and (5S,6S)-5-hydroxy-6-methylpiperidin-2-one, and the former intermediate was successfully used in the synthesis of (+)-2-epi-deoxoprosopinine and ()-deoxoprosophylline. For the synthesis of cis-DHQ alkaloids, (4aR,5S,8aS)- and (4aR,5R,8aS)-5-[(phenylsulfonyl)methyl]octahydroquinolin-2-ones were prepared by employing a 6-exo-trig free-radical cyclization reaction as the key step. The stereoselectivity observed in the free-radical cyclization step was found to be governed by allylic 1,2-strain arising from the interaction of the N-(p-methoxybenzyl) group and the C6 substituent in the lactam ring of the free-radical intermediate. The (4aR,5S,8aS)- and (4aR,5R,8aS)-5-[(phenylsulfonyl)methyl]octahydroquinolin-2-ones were successfully converted to (+)-cis-195A and 2,5-diepi-cis-195A, respectively. Further, a competitive oxidation pathway was uncovered in the cerium(IV) ammonium nitrate oxidation of N-PMB -lactams, which resulted in the unprecedented formation of N-(hydroxymethyl) -lactam along with the desired unprotected lactam. A mechanistic rationalization for this unexpected outcome was proposed.
Open Access
Targeted knockout of PpASCL, an anther-specific chalcone synthase-like gene, supports role in sporopollenin biosynthesis in the moss Physcomitrella patens.
(Faculty of Graduate Studies and Research, University of Regina, 2014-09) Daku, Rhys Michael; Suh, Dae-Yeon; Ashton, Neil; Dahms, Tanya; Weger, Harold
The anther-specific chalcone synthase-like (ASCL) gene of Physcomitrella patens, PpASCL, has been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. Targeted knockouts of PpASCL were produced and the resulting mutants characterized. Knockout plants developed normally until late in sporophytic development, when the spores produced were aberrant and non-viable. The development of the spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with irregular surface morphology. These results suggest that PpASCL plays an important role in the proper formation of the exine layer, and is required for the maturation and viability of moss spores.
Open Access
Targeted knockout of PpORS encoding an ancient type III polyketide synthase in the moss Physcomitrella patens
(Faculty of Graduate Studies and Research, University of Regina, 2015-12) Li, Li; Suh, Dae-Yeon; Ashton, Neil; Dahms, Tanya
The ubiquitous presence of type III polyketide synthases (PKS) in the plant kingdom suggests their important roles in plant evolution. Among plant type III PKSs, PpORS from the model moss, Physcomitrella patens, has previously been suggested to closely resemble the most recent common ancestor of plant type III PKSs. In vitro analysis of PpORS revealed its function as a 2'-oxoalkylresorcinol synthase. PpORS is highly expressed in gametophores, but not in protonema. This, together with previous phytochemical analysis, suggested that in planta PpORS products may be incorporated into the moss cuticular structure to confer protection from environmental stresses. In this study, to gain insight into the in planta function of PpORS, PpORS was targeted for knockout in Physcomitrella. Three stable transformants were confirmed by PCR, Southern blot and RT-PCR, and PpORS stable transformants (ors) were phenotypically analyzed. Ors plants were similar to the wild-type plant in developmental characteristics, including branch numbers and spore viability. Both the wild-type and ors plants responded similarly to UV-B irradiation, suggesting that UV protection is not a main function of PpORS. In contrast, abnormal leaves, including (i) curly leaves with a large number of protuberances, (ii) highly deformed leaves, (iii) irregularly shaped leaves, and (iv) leaves with cell outgrowths on the surface or margin, were frequently observed in ors. Abnormal leaves were heavily stained by a hydrophilic dye Toluidine Blue O, whereas both normal and abnormal leaves were stained by Sudan IV. Interestingly, the incidence of morphological abnormalities in ors leaves significantly decreased with water immersion treatment. These results suggest that ors shoots may be covered with a defective cuticle layer.
Open Access
Targeted Knockouts of Selected GDSL Esterases and Class III Peroxidases In The Moss Physcomitrella patens Reveal Their Roles in Spore Wall Formation and Germination
(Faculty of Graduate Studies and Research, University of Regina, 2020-06) Rabbi, Fazle; Suh, Dae-Yeon; Dahms, Tanya; Weger, Harold; Ashton, Neil; Singer, Stacy
Early land plants evolved a number of key innovations to invade the land and diversify. One such innovation was a robust spore wall containing sporopollenin. Sporopollenin is the main lipidic component of spore and pollen walls and has been proposed to be the decisive factor for the successful terrestrialization by early plants. Until now, no enzyme involved in sporopollenin metabolism (polymerization and degradation) has been studied at the genetic and biochemical levels. I hypothesized that sporopollenin metabolizing enzymes might be homologs of cutin and lignin metabolizing enzymes belonging to GDSL esterases and class III peroxidase families, respectively. In this study, two GDSL esterases (CUSL and CUTL) and three class III peroxidases (PRX38, PRX39 and PRX47) of a model plant, Physcomitrella patens, were selected as candidate enzymes in sporopollenin metabolism based on gene expression profiles and phylogenetic analysis. Targeted knockout experiments were performed, and stable single (cutl, cusl and prx47) and double knockout (prx38 prx39) plants were generated. Stable knockout lines were confirmed by gDNA PCR. Single targeted gene replacement in the cutl and cusl lines was confirmed by Southern blot and RT-PCR. Then the stable knockout lines were phenotypically analyzed. Cutl and prx47 spores showed delayed germination, whereas early germination was observed with cusl spores as compared to control spores. In addition, cusl and prx38 prx39 spores showed augmented osmolysis and cusl spores were more susceptible to alkaline hydrolysis than control spores. These results suggest that we may have discovered novel GDSL esterases and class III peroxidases involved in spore wall formation (CUSL, PRX38 and/or PRX39) and degradation (CUTL and PRX47) in Physcomitrella. In addition, treatment of developing sporophytes with ROS scavengers provided support for the involvement of oxidative cross-linking in spore wall development, including sporopollenin polymerization and/or deposition, as well as a role for ROS in intine/aperture development. This research contributes to our understanding of the metabolism of sporopollenin and its evolution in land plants, and may have direct applications involving this biopolymer in the fields of biomedicine, biotechnology and green chemistry.
Open Access
The polyketide pathway in sporopollenin biosynthesis is specific to land plants (Embryophyta)
(BioRxiv, 2024-10-19) Sraan, Damanpreet K.; Ashton, Neil W.; Suh, Dae-Yeon
Background and Aims: Sporopollenin (SP) is a complex biopolymer in the outer wall of spores and pollen and provides protection from environmental stresses. Its extraordinary chemical resistance, especially to acetolysis, was widely used to identify SP in biological specimens. This broad definition of SP led to claims for its widespread occurrence among diverse embryophyte and non-embryophyte taxa. We previously proposed a biochemical definition that can be used to distinguish genuine SP from other chemically resistant cell wall materials. The definition was centred on ASCL (Anther-Specific Chalcone synthase-Like), an embryophyte-specific enzyme of the polyketide pathway that provides precursors for SP biosynthesis. Herein, we examine the evolution and distribution of all five enzymes (CYP703A, CYP704B, ACOS, ASCL and TKPR) of the polyketide pathway and propose a new, more comprehensive definition of SP. Methods: We performed BLASTp searches, phylogenetic tree construction, protein modeling and sequence analysis to determine the presence or absence of ACOS and TKPR in embryophytes and streptophytic algae. Key Results: We found evidence that all five enzymes of the polyketide pathway evolved from ancestral enzymes of primary metabolism and ACOS, ASCL and TKPR were co-selected during evolution. The dosage of all five genes has been subjected to strict evolutionary control and, in some taxa, synteny has provided a selective advantage. All five enzymes are present in embryophytes but absent in green algae, indicating that the polyketide pathway and therefore SP is embryophyte-specific. Conclusions: The addition of the polyketide pathway in the definition of genuine SP will allow separation of SP from algaenans and other chemically resistant ‘SP-like’ algal spore wall substances. This study further signifies SP as an evolutionary innovation unique to the embryophyte lineage and encourages research on possible evolutionary relationship between algal spore wall ‘SP-like materials’ and embryophyte SP. Competing Interest Statement The authors have declared no competing interest.