Browsing by Author "Chao, Tzu-Chiao"

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Open Access
Alzheimer's Disease and Amyloid Beta: Effects of amyloid beta on hippocampal and dorsal root ganglion neurons' electrophyiology and morphology
(Faculty of Graduate Studies and Research, University of Regina, 2019-07) Castilla Bolanos, Maria Alejandra; Buttigieg, Josef; Chao, Tzu-Chiao; Mousseau, Darrell; Campanucci, Veronica
Alzheimer’s disease (AD) is the most common type of dementia, increasing by 10 new cases every day in Saskatchewan alone. AD results from pathological amyloid beta (Aβ) peptides that hamper neurons’ communication, cause progressive neuronal cell death in the central nervous system, and ultimately lead to memory loss in the individual. While disease symptoms are well known, the mechanism by which Aβ triggers the degeneration of the brain remains elusive. There is no cure for AD and present treatments only focus on slowing down the disease progression or mitigating symptoms. This study has used several techniques to investigate the effect of Aβ on the electrophysiology and morphology of isolated hippocampal and dorsal root ganglion (DRG) neurons in order to test whether the effects of Aβ exposure and clearance are exclusive to central nervous system neurons. Isolation and culture of hippocampal and DRG neurons were performed. By exposing neurons to combinations of Aβ42 and Aβ38, electrophysiological and morphological techniques were used to assess the effects of Aβ on neuronal cells. Using patch clamp electrophysiology, I demonstrated a significant decrease in the current density profile of neurons after 24-h of exposure to Aβ42 and Aβ38. Additionally, Aβ had a dose-dependent effect on hippocampal and DRG neurons’ morphology, reducing neuronal soma and nucleus size. The addition of Aβ38 negated the depressive effect of Aβ42, suggesting the cancellation of low current density profiles of neurons or the inhibition of their electrical activity. Thus, co-treatment of Aβ42 and Aβ38 neutralized the depressive Aβ effect on neuronal cells. This research has demonstrated the electrophysiology of hippocampal and DRG neurons after Aβ exposure to understand the fundamental biology of Aβ exposure at a cellular level, which is involved in the pathophysiology of AD and other types of dementia.
Open Access
Blocks in Tricarboxylic Acid Cycle of Salmonella enterica Cause Global Perturbation of Carbon Storage, Motility, and Host-Pathogen Interaction
(American Society for Microbiology, 2019-12-11) Noster, Janina; Hansmeier, Nicole; Persicke, Marcus; Chao, Tzu-Chiao; Kurre, Rainer; Popp, Jasmin; Liss, Viktoria; Reuter, Tatjana; Hensel, Michael
The tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (S. Typhimurium) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in the TCA cycle, a strain deficient in all fumarase isoforms (ΔfumABC) elicited a unique metabolic profile. Alongside fumarate, S. Typhimurium ΔfumABC accumulates intermediates of the glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes toward glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of S. Typhimurium by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis and partially restored levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential- to stationary-growth-phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of S. Typhimurium and cellular functions and virulence.
Open Access
Characterization of the Microbial Communities Within Managed Bioremediation Systems: A Culture- and Genomics-Based Approach
(Faculty of Graduate Studies and Research, University of Regina, 2020-12) Russell, Jennifer Nadine; Dahms, Tanya; Chao, Tzu-Chiao; Hartmann, Erica
Wastewater treatment facilities (WWTF) and biobeds are engineered systems developed to mitigate nutrient and pesticide pollution, respectively, through microbial biodegradation. While understanding the microbiomes of these systems may help inform on optimization and maintenance, genomic characterization of these systems is generally lacking, particularly in reference to biobeds, with many questions regarding which microbes are present, and how these microbial communities change across the treatment process over time. Within this, an additional point of interest relates to the unintended negative effects these systems might pose, specifically as potential environments for the enrichment of antibiotic resistance genes (ARGs). WWTFs have become known hubs for ARG dissemination, with studies reporting increases in both ARGs and antibiotic resistant (ABR) bacteria in effluentreceiving water systems. Here, through culturing, it was identified that the recently upgraded Regina Wastewater Treatment Plant is capable of reducing bacterial populations by 95-99 %, but that resistance to ampicillin, trimethoprim and meropenem is high, and that ABR bacteria are surviving the treatment process. Additionally, through 16S rRNA and cpn60 amplicon sequencing, a significant change in community composition was observed over the treatment continuum, which is most likely driven by significant decreases in diversity, driven through UV disinfection, although results suggest that UV treatment may be selecting for bacteria housing trimethoprim-resistance genes. Finally, multiple multidrug resistant (MDR) Gammaproteobacteria were observed to be surviving the wastewater treatment continuum, and are being released into the Qu’Appelle Valley watershed, indicating that there may be potential for ABR dissemination through this aquatic ecosystem. Lastly, through studying the microbiome of this wastewater system, the sequencing results of the two different amplicon targets – 16S rRNA and cpn60 – were compared. Results suggest a high incidence of primer bias, and indicated that the results provided from these two methods are capable of arriving at different conclusions regarding taxonomy and statistical significance. Biobeds were studied through the context of taxonomy, degradative genes, and also their potential for ARG enrichment through metagenomic and metatranscriptomic sequencing of the Lethbridge Biobed system. Results show that pesticides enrich for bacteria commonly associated with xenobiotic degradation, such as Afipia, Sphingopyxis and Pseudomonas, and enrich for xenobiotic-degrading genes, such as peroxidases, oxygenases, and hydroxylases, among others; the transcription of these genes was able to be directly linked to Pseudomonas, Oligotropha, Mesorhizobium, Rhodopseudomonas, and Stenotrophomonas taxa. Sequencing analyses further demonstrated an absence of ligninolytic fungi, which is contrary to other microbially characterized biobeds, suggesting that biobeds are highly variable in their microbial structure, and that biobeds can function effectively in the absence of fungi. Finally, because ABR selection and enrichment is driven by increased stress, which leads to increased rates of horizontal gene transfer and ARG acquisition, genes that are related to stress response systems, as well as genetic recombination were statistically analyzed alongside ARGs. It was found that the pesticide concentrations being applied to a biobed was high enough to enrich for, and transcribe, genes related to a variety of stress responses, such as repair systems and metabolism modulation, genetic recombination, such as conjugation and plasmids, and ARGs, such as aminoglycoside and MDR genes, showing that biobed systems may need to be monitored for ABR dissemination.
Open Access
Deciphering Metabolic Adaptations in Salmonella Enterica Serovar Typhimurium 14028S During Batch Growth and Gentamicin Exposure Using Absolute Quantitative Proteomics
(Faculty of Graduate Studies and Research, University of Regina, 2022-01) Sharma, Samrachana; Chao, Tzu-Chiao; Cameron, Andrew; Sterzenbach, Torsten
Understanding the mechanisms underlying antibiotic-mediated bacterial cell death is crucial for the development of novel strategies to control bacterial infections. Although the primary targets of antibiotics are well known, the actual molecular events leading to bacterial growth inhibition and cell death remain unclear. Recent studies have suggested that the interaction of antibiotics with their primary targets in bacteria results in harmful events, such as futile cycles, activation of toxic pathways, and accumulation of harmful by-products that cause bacterial growth inhibition and death. However, these events remain only partially understood and evidence suggests an intricate interplay between metabolic activities of the bacterium and the perturbations caused by antibiotics. To unravel this interplay, global analyses of bacterial responses to antibiotic treatment are required. In this thesis, we performed absolute quantification of the proteome in the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) using data-independent acquisition (DIA) mass spectrometry (MS)-based approach. We first mapped the metabolic activities of S. Typhimurium cells throughout batch growth (early, mid, and late logarithmic and early stationary phase) in order to establish a baseline of metabolic states during regular unperturbed growth. Our data show that the logarithmic growth is not uniform due to metabolic shift as a result of continuous depletion of the medium. For instance, we observed a reduced abundance of proteins involved in translation within the logarithmic phases (mid and late logarithmic) corresponding to a reduced growth rate. Moreover, the proteomic profiles suggested a switch from glycolysis to gluconeogenesis from the mid-logarithmic phase (4.5 h) onwards, likely the result of glucose depletion in the medium. We also observed an increase in proteins involved in amino acid metabolism (Asp, Gly, Ser, Thr) beyond the mid-logarithmic phase. Although these amino acids could fuel the TCA cycle to provide precursors for gluconeogenesis, the proteome data does not conclusively indicate either anabolic or catabolic pathways. In presence of gentamicin, we observed an increase in the abundance of ribosomal proteins, suggesting that S. Typhimurium might compensate for ribosomal inhibition by synthesizing additional ribosomes. Moreover, we observed an increased abundance of proteins involved in multiple metabolic pathways such as fatty acid metabolism and oxidative phosphorylation during gentamicin exposure, suggesting that the ribosomal inhibition by gentamicin can result in pleiotropic metabolic effects. Furthermore, we demonstrated that the addition of glucose improves the ability of S. Typhimurium to grow in presence of gentamicin. Proteomic profiling revealed that after supplementation of glucose the abundance of proteins involved in glycolytic and fermentative pathways increased, whereas proteins of the TCA cycle were reduced in abundance. Previous studies suggested that bacterial susceptibility towards other ribosome-targeting antibiotics may be related to the TCA cycle. Based on our data, we speculate that if enough energy can be generated from glycolytic pathways, bypassing the TCA cycle could support S. Typhimurium survival in the presence of gentamicin.
Open Access
Effect of Physioxia on Protein Levels in Adult Mouse (Mus Musculus) Neural Stem and Progenitor Cells
(Faculty of Graduate Studies and Research, University of Regina, 2018-12) Lanigan, Adam Thomas; Manzon, Richard; Chao, Tzu-Chiao; Yost, Christopher; Babu, Mohan
Neural Stem and Progenitor Cells (NSPCs) from the adult subventricular zone hold therapeutic potential for a variety of neurological diseases due to their capabilities of self-renewal and potential for differentiation into the cell types of the brain. The potential for autologous cell replacement using NSPCs in cases of neurological injury or disease alleviates many of the ethical and practical issues that are a concern of other stem cell varieties. However, an understanding of many of the basic mechanisms that influence NSPC behaviour under normal physiological conditions is still required to develop appropriate strategies for NSPC expansion in vitro or for endogenous recruitment. Typically studies of NSPCs are conducted under conditions of atmospheric O2 levels, when in reality NSPCs reside in a niche that is subject to much lower concentrations of O2. As such, most studies concerning NSPCs may be limited in terms of their physiological relevance, and culture at lower levels of O2 may better approximate the conditions NSPCs experience in situ within their neurological niche. With this in mind, adult mouse (Mus musculus) NSPCs were cultured in both physiologically relevant levels of O2 (5%) and atmospheric conditions (21% O2) and then subsequently harvested for quantitative proteomic analysis using a data-independent acquisition (DIA) mass spectrometry (MS)-based approach. While cell count experiments suggested there was a difference in cellular activity between NSPCs in each treatment, there were no statistically significant changes in individual protein levels between O2 treatment groups following MS analysis, likely due to high variation among samples. However, MSanalysis still resulted in the identification of 878 unique proteins across treatment groups with quantitative protein expression values. To the best of our knowledge, this is the first quantitative accounting of the proteome of adult NSPCs from mice using DIA-MS-based techniques, which has resulted in a data set that can be compared to future studies of NSPCs. Additionally, this paper provides a framework for future studies by implementing coarse-grain functional categorization of proteins in order to reduce a large proteomic data set into a partitioning scheme that may be more amenable to deriving biologically meaningful conclusions.
Open Access
Establishing wastewater surveillance for SARS-CoV-2 in Southern Saskatchewan
(Faculty of Graduate Studies and Research, University of Regina, 2023-06) Reuter, Sonja Tamara; Hansmeier, Nicole; Chao, Tzu-Chiao; Yost, Christopher; Van Hamme, Jonathan
Wastewater surveillance has become a crucial part in the monitoring of the COVID-19 pandemic. Infected people shed SARS-CoV-2 in their feces, therefore, virus levels in wastewater reflect the trends in infection numbers in the population that contributes to the wastewater. Wastewater surveillance offers information about the spread of SARS-CoV-2 independent of testing strategies and individual choices and therefore better reflects population health than individual testing. In this study we compared five RNA extraction kits and reagents and different enrichment methods for wastewater surveillance of SARS-CoV-2 RNA and determined that the solids fraction of wastewater was most suitable for RNA extraction using the AllPrep PowerViral DNA/RNA Kit by Qiagen. We also examined the impact of storage on wastewater samples and determined that while short-term storage does not affect the samples significantly, longer storage changes the measured viral levels. We then used our protocol to establish a wastewater surveillance for SARS-CoV-2 viral levels in Regina and nine other locations in Southern Saskatchewan, Canada. We observed that the three waves of COVID-19 during the study time were caused by the variants Alpha, Delta and Omicron and that the SARSCoV- 2 RNA levels in wastewater were a good reflection of the reported active COVID-19 case numbers.
Open Access
Functional genomics provides insights into the genetic network(s) associated with the function of the cmd operon in Rhizobium leguminosarum bv. viciae
(Faculty of Graduate Studies and Research, University of Regina, 2020-08) Akter, Mir Salma; Yost, Christopher; Sugh, Dae-Yeon; Cameron, Andrew; Chao, Tzu-Chiao; Cardona, Silvia
The bacterium Rhizobium leguminosarum forms a symbiotic association with leguminous plants such as peas and lentils, which are important food crops. Rhizobium’s cell envelope is important for establishing a symbiotic association with host plants and for helping the cell adapt to stressful conditions in the rhizosphere. A previously uncharacterized four gene operon, cmdA-D (RL3499-RL3502) in Rhizobium leguminosarum viciae 3841 has been shown to be essential for proper cell envelope function. The broadly conserved cmd operon codes for ATPases which mediate protein-protein interaction and are involved in various cellular activities. cmd mutants cannot grow on complex peptide rich media, therefore the operon was named for the complex media deficient (cmd) phenotype of the mutants. However, cmd mutants can grow on minimal media. Apart from the inability to grow on peptide rich media (TY), these mutants exhibit some additional phenotypes such as distorted enlarged cell shape, sensitivity to cell envelope stressors like detergents, hydrophobic antibiotics, alkaline pH, and production of twice as many nodules in pea plants compared to the wild type. At low frequencies, strains that are able to grow on peptide rich TY media, can be isolated from the cmd operon mutants. These isolates represent secondary site mutations that are supressing the original cmd mutant phenotype. One such isolate 38cmdB_S, able to grow on TY, isolated from a cmd mutant in non-polar RL3500 (38cmdB) mutant background was selected for further characterization. This study aims to increase our understanding of the biological function of the cmd operon by studying the 38cmdB mutant and the suppressor mutant, 38cmdB_S through phenotypic characterisation, genome wide mutagenesis using insertion sequencing (INSeq), and transcriptomic analysis through RNA-Seq of the wild type, 38cmdB and 38cmdB_S mutant. ii INSeq, a high throughput genetic screening technique was used to simultaneously identify the suppressor locus and to identify conditional essential genes in the suppressor mutant background. Using this approach and selection on TY media, 40 conditional essential genes were found in the suppressor mutant. The Insertion sequencing data not only reveals the putative suppressor locus but also other genes which are connected to the function of the cmd operon. Metal chelatase, cell envelope biosynthesis proteins, genes involved in ABC transporter, and in glycolysis and gluconeogenesis were identified as necessary to compensate for the loss of cmd operon’s function in the suppressor strain. In this study, it was discovered that the 38cmdB mutant could regain growth on TY if the culture medium was supplemented with metals (magnesium, iron, calcium) and vitamins (biotin, thiamine, pantothenic acid), indicating the necessity of high concentration of metals and vitamins in the absence of functional cmd operon. Moreover, the comparative transcriptomic data (for the same growth condition) between wild type, and both the cmdB mutant and the suppressor mutant added new information about the genetic network associated with the cmd operon. Genes involved in siderophore synthesis, cell wall biosynthesis, and different transporters such as ABC, MFS, and RND were observed to be upregulated in both mutant strains, suggesting changes in gene regulation in both mutants associated with the loss of cmdB (RL3500) gene and cmd operon’s function. The exact molecular function of the cmd operon is still unknown, however the involvement of cmd operon in protein maturation, in insertion of divalent metals into other proteins, and in maintaining the cell envelope integrity during polar growth in Rhizobium leguminosarum can be suggested from this study.
Open Access
Genetic mechanisms contributing to antimicrobial resistance: gene repression in Mycobacterium, gene transfer in Salmonella, and phage sensitivity in Escherichia
(Faculty of Graduate Studies and Research, University of Regina, 2018-07) Yoneda, Joshua Raj Kotaro; Cameron, Andrew; Chao, Tzu-Chiao; Dahms, Tanya
Bacteria develop resistance to antimicrobial agents through genetic mutation or horizontal acquisition of genetic material from resistant cells. Mycobacterium, Salmonella, and Escherichia are all capable of causing disease, and each demonstrates increasing resistance to conventional antibiotic treatment. Understanding the genetic mechanisms that regulate antibiotic resistance are critical in developing new antimicrobials and improving current treatment regimens. Alternative therapies to antibiotics should also be considered in the fight against multidrug resistant pathogens. Thus, I carried out molecular studies to investigate the regulation of efflux pump expression in Mycobacterium intracellulare, regulation mechanisms of natural competence in Salmonella Typhimurium, and phage therapy of Enterohemorrhagic Escherichia coli serogroup O157:H7. A recent study showed specific mutations in the uncharacterized locus mmpT5 increased resistance to anti-TB drug bedaquiline in M. intracellulare lung infections. Based on previous work, MmpT5 is a TetR transcription factor hypothesized to repress expression of the downstream Resistance Nodulation Division (RND) efflux pump mmpSL. To test this, we assembled multigene constructs on a plasmid with the modular cloning (MoClo) toolkit, which allowed us to control the expression of mmpT5 with the lacZ promoter and monitor mmpSL promoter activity with a fusion to lux. Consistent with the hypothesis, induction of mmpT5 reduced expression of mmpSL. The nonsynonymous mutations in mmpT5 first identified in clinical isolates resulted in upregulation of mmpSL, suggesting a mechanism for bedaquiline resistance. The periplasmic protein ComA is essential for active uptake of DNA from the environment. In Salmonella Typhimurium, comA is transcriptionally silent, even when the predicted activator Sxy is overexpressed. Identification of a mRNA stem-loop in the comA promoter region provides a possible gene repression mechanism. We used the MoClo toolkit to assemble comA promoter-luciferase fusions with or without the predicted mRNA secondary structure, and we used site-directed mutagenesis (SDM) to destabilize the predicted mRNA stem-loop. Luciferase reporter assays showed stem-loop removal or destabilization via SDM increased comA expression under the control of the lacZ promoter. SDM destabilization of the mRNA stem-loop did not increase luminescence of the native comA, but induction of sxy resulted in a 197-fold increase in luminescence compared to the wild type when the predicted stem-loop was destabilized, presenting a clear explanation for the cryptic nature of comA expression in Salmonella. Phage therapy presents an attractive alternative to conventional antibiotic treatment, prompting a study of the sensitivity of Enterohemorrhagic Escherichia coli (EHEC) serogroup O157:H7 to phage killing using a tetrazolium reduction assay. Measuring tetrazolium reduction at A485 successfully showed which EHEC strains were sensitive to the T4-like typing phage 13 (TP13). Addition of T7-like phage TP9 to TP13 increased the efficacy of phage treatment. TP13 resistance was observed in EHEC strains with the stx2a lysogen. RT-qPCR and plasmid complementation experiments showed that the hypothetical gene H2 was not responsible for TP13 resistance. Subsequent review of recent literature revealed that the downstream tyrosine kinase stk is likely responsible for TP13 resistance through abortive infection. Altogether, this thesis studies three bacterial systems to better understand the genetic bases of antimicrobial resistance, including gene transfer mechanisms, and to find the genetic bases of phage sensitivity.
Open Access
Impact of ROS-Induced Damage of TCA Cycle Enzymes on Metabolism and Virulence of Salmonella enterica serovar Typhimurium
(Frontiers Media, 2019-04-24) Noster, Janina; Persicke, Marcus; Chao, Tzu-Chiao; Krone, Lena; Heppner, Bianca; Hensel, Michael; Hansmeier, Nicole
Salmonella enterica serovar Typhimurium (STM) is exposed to reactive oxygen species (ROS) originating from aerobic respiration, antibiotic treatment, and the oxidative burst occurring inside the Salmonella-containing vacuole (SCV) within host cells. ROS damage cellular compounds, thereby impairing bacterial viability and inducing cell death. Proteins containing iron–sulfur (Fe–S) clusters are particularly sensitive and become non-functional upon oxidation. Comprising five enzymes with Fe–S clusters, the TCA cycle is a pathway most sensitive toward ROS. To test the impact of ROS-mediated metabolic perturbations on bacterial physiology, we analyzed the proteomic and metabolic profile of STM deficient in both cytosolic superoxide dismutases (ΔsodAB). Incapable of detoxifying superoxide anions (SOA), endogenously generated SOA accumulate during growth. ΔsodAB showed reduced abundance of aconitases, leading to a metabolic profile similar to that of an aconitase-deficient strain (ΔacnAB). Furthermore, we determined a decreased expression of acnA in STM ΔsodAB. While intracellular proliferation in RAW264.7 macrophages and survival of methyl viologen treatment were not reduced for STM ΔacnAB, proteomic profiling revealed enhanced stress response. We conclude that ROS-mediated reduced expression and damage of aconitase does not impair bacterial viability or virulence, but might increase ROS amounts in STM, which reinforces the bactericidal effects of antibiotic treatment and immune responses of the host.
Open Access
Prioritization of biomarker targets in human umbilical cord blood: identification of proteins in infant blood serving as validated biomarkers in adults
(National Institute of Environmental Health Sciences (NIEHS), 2012-05-01) Hansmeier, Nicole; Chao, Tzu-Chiao; Goldman, Lynn R; Witter, Frank R; Halden, Rolf U
Early diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped.
Open Access
Proteomic Analysis of Salmonella-modified Membranes Reveals Adaptations to Macrophage Hosts*
(American Society for Biochemistry and Molecular Biology (ASBMB), 2020-05) Reuter, Tatjana; Vorwerk, Stephanie; Liss, Viktoria; Chao, Tzu-Chiao; Hensel, Michael; Hansmeier, Nicole
Systemic infection and proliferation of intracellular pathogens require the biogenesis of a growth-stimulating compartment. The gastrointestinal pathogen Salmonella enterica commonly forms highly dynamic and extensive tubular membrane compartments built from Salmonella-modified membranes (SMMs) in diverse host cells. Although the general mechanism involved in the formation of replication-permissive compartments of S. enterica is well researched, much less is known regarding specific adaptations to different host cell types. Using an affinity-based proteome approach, we explored the composition of SMMs in murine macrophages. The systematic characterization provides a broader landscape of host players to the maturation of Salmonella-containing compartments and reveals core host elements targeted by Salmonella in macrophages as well as epithelial cells. However, we also identified subtle host specific adaptations. Some of these observations, such as the differential involvement of the COPII system, Rab GTPases 2A, 8B, 11 and ER transport proteins Sec61 and Sec22B may explain cell line-dependent variations in the pathophysiology of Salmonella infections. In summary, our system-wide approach demonstrates a hitherto underappreciated impact of the host cell type in the formation of intracellular compartments by Salmonella.
Open Access
Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense
(Public Library of Science, 2019-04-22) Noster, Janina; Chao, Tzu-Chiao; Sander, Nathalie; Schulte, Marc; Reuter, Tatjana; Hansmeier, Nicole; Hensel, Michael
Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella-induced filaments (SIFs) connected to the Salmonella-containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as ΔssaV. A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), ΔsseF or ΔssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM ΔsseF and ΔssaV compared to WT. In addition, STM ΔssaV, but not ΔsseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms.
Open Access
Role of the Neurotransmitter Glutamate in Neural Stem and Progenitor Cells Differentiation into Oligodendrocytes
(Faculty of Graduate Studies and Research, University of Regina, 2017-12) Kisheev, Anastasye; Buttigieg, Josef; Chao, Tzu-Chiao; Dahms, Tanya
Various regions of the mammalian brain, including the subventricular zones (SVZs) of the hippocampus and the dentate gyrus (DG), contain niches of undifferentiated neural stem and progenitor cells (NSPCs) which persist from development, all throughout adulthood. These cells can differentiate into the three main cell types found within the brain: neurons, oligodendrocytes (OLs), and astrocytes. Adult NSPCs can be driven to specific cells of interest which can be used to replace lost myelin in some cases of neurodegenerative diseases such as multiple sclerosis (MS) or insults such as spinal cord injury. I investigated the requirements to drive immature NSPCs into oligodendrocyte precursor cells (OPCs) using various driving factors (PDGFα and NT-3) and the neurotransmitter (NT) glutamate. Glutamate is one of the main NTs in the central nervous system (CNS) hence, it may have a role in the differentiation of NSPCs into OPCs. My results demonstrated that among all the driving factors tested, glutamate treatment yielded the highest proportion of OPCs after six days. Flow cytometry was used to verify changes in cell lineage and proportions during the driving experiments. Enriched OPC cultures were purified using a magnetic bead sorting technique after which the cell lineage was confirmed using an immunocytochemistry (ICC) staining technique. Results demonstrated that cells differentiated alone with no external stimulation did not produce the myelin basic protein (MBP). In contrast, almost all OPCs stimulated with glutamate expressed MBP. Purified OPCs grown in a co-culture with DRG neurons for nine days also began expressing MBP which was found to be in close association with the neuronal axons. In order to verify the effect of glutamate in co-cultures of OPCs with DRG neurons, AMPA/kainate inhibitors such as CNQX and DNQX, were tested. Although MBP positive cells were still identified in cultures treated with AMPA inhibitors, in the case of CNQX, the myelin sheath organization appeared distorted compared to the nontreated samples, thus suggesting glutamate does play a key role in oligodendroglial development.
Open Access
The role of neurosteriod fluctuation in perimenopausal depression
(Faculty of Graduate Studies and Research, University of Regina, 2023-08) Sander, Bethany Jane; Gordon, Jennifer; Wright, Kristi; Chao, Tzu-Chiao
There is a well-established increase in depression risk during the menopause transition – known as perimenopausal depression. Increased fluctuation of estradiol has long been thought to be an important causal factor in perimenopausal depression, and recent research suggests that increases, decreases, or absolute changes in estradiol may be associated with depressive symptoms in different subsets of perimenopausal women. However, the mechanisms by which estradiol differentially influences mood are still very unclear. Preliminary evidence suggests that estradiol may increase vulnerability to developing depressive symptoms through the effects of a neurosteroid called allopregnanolone. In the current study, the role of estradiol and allopregnanolone was examined in 26 perimenopausal participants experiencing disturbed mood. Fifteen weekly measurements of depressive symptoms as well as plasma levels of estradiol, progesterone, and allopregnanolone were collected. In 10 participants with a sufficient number of weeks of hormone data available, correlational analysis revealed that every participant displayed a significant relationship between depressive symptoms and changes in at least one neurosteroid. Although mood sensitivity to progesterone was correlated with mood sensitivity to allopregnanolone, estradiol and allopregnanolone sensitivity indicators appeared not to be related. Our results suggest that fluctuations in neurosteroids in the menopause transition play an important role in perimenopausal depressed mood. Future research is needed to clarify whether estradiol and allopregnanolone changes each play an independent role in exerting effects on perimenopausal mood or work in tandem.