Browsing by Author "Harrison, Joe J."
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Item Open Access Comparing the mechanisms of metal action in bacteria: insight into novel genes involved in silver, gallium and copper resistance and toxicity in Escherichia coli(2019-07-25) Gugala, Natalie; Turner, Raymond Joseph; Harrison, Joe J.; Lewis, Ian A.; Shimizu, George K. H.; Mulvey, MichaelIt is fundamental to understand the mechanisms by which a toxicant is capable of poisoning the bacterial cell or resistance is developed. The mechanisms of actions of many antimicrobials such as metal-based compounds are not fully understood, yet, the development of these agents continues. Despite the essentiality of metals in the biochemistry of life, both non-essential and essential metals have been used as antimicrobials for agricultural and medical purposes for thousands of years. Applications include wound dressings, nanoparticles, antiseptic formulations, combination treatments, polymers and nanocomposites, among many more. Many of these have proven to be effective at controlling and eradicating microbial populations at low concentrations. Currently, studies in this field largely focus attention on developing new formulations and utilities for metal-based antimicrobials. The identity of the cellular targets that are involved in metal resistance and toxicity are known to a lesser degree. This current knowledge gap potentiates the progression of antimicrobial resistance since there is an incomplete understanding of metal action in microorganisms. Previous studies that have directed efforts toward these fundamental questions have failed to provide a comprehensive depiction of the global cellular effects of metal exposure; the literature is often replete with contradicting reports. Based on the aforementioned, we sought to answer the fundamental question – how do the mechanisms of metal toxicity and resistance compare in bacteria? We observed that the efficacies of metal ions varied between bacterial species and isolates of the same species. By means of the Keio collection, this comparison was validated by demonstrating that silver, copper and gallium act differently in Escherichia coli. Here, we presented a list of novel resistant and sensitive gene hits that may be involved in metal action. These experiments were performed under sublethal prolonged metal exposure, rather than acute shock. Resistance mechanisms range from efflux, iron-sulfur cluster maintenance, DNA repair, nucleotide biosynthesis to tRNA modification, and sensitive pathways include biomolecule import, NAD+ synthesis, amino acid biosynthesis, sulfur assimilation, electron transport, carbon metabolism and outer membrane maintenance, amongst others. To mitigate the improper use of metal-based antimicrobials, it is imperative that we understand precisely how these agents are able to eradicate bacterial cells and what are the accompanying mechanisms of resistance, particularly as development and use expands.Item Open Access Conjugative Complexities: Defining the requirements for the transfer of Rhizobium leguminosarum plasmid pRleVF39b(2019-03-20) Wathugala, Nandun Dulmini; Hynes, Michael F.; Turner, Raymond Joseph; Harrison, Joe J.The genome of Rhizobium leguminosarum is compartmentalized into a chromosome and multiple large (> 100 kb) plasmids. Four conjugation systems have been identified that can mediate horizontal transfer of these plasmids, and type IVA-1 was identified on R. leguminosarum VF39SM plasmid pRleVF39b. This category of system is found among multiple Rhizobiaceae members from different geographical locations, on both non-symbiotic and symbiotic plasmids. The type IVA-1 conjugation system on pRleVF39b is distinctive due to the presence of a shorter relaxase gene (traA) compared to canonical relaxases, a repressor, TrbR, belonging to the xenobiotic response element (Xre)-family, and the lack of some common genes found in the other conjugation systems. There are 15 annotated Orfs adjacent to known transfer genes in the type IV conjugation locus that might have important functions in plasmid transfer. A series of deletions in each of the hypothetical genes was created to identify essential and facilitating genes for the transfer of pRleVF39b. The effect of these mutations on transfer of pRleVF39b to plasmid-free Agrobacterium recipients was analyzed. orf23, orf24, orf26, orf27, orf28 and orf29 mutants reduced the plasmid transfer frequency by 10-fold or more compared to wild type, suggesting a key role in conjugation. To define the TrbR regulon of pRleVF39b, putative promoter (P) regions of orf17, trbR, orf24, orf25, orf26 and orf29 were fused to a reporter gene gusA. The expression of promoters in WT VF39, VF39a-, VF39b-, trbR mutant and an orf29 mutant was analyzed using β-glucuronidase assays. PtrbR expression was not reduced due to trbR mutation. Thus, TrbR does not autoregulate itself. P24 showed ~30-fold increase and P25 showed ~10-fold increase in expression in VF39b- and trbR backgrounds, indicating that the operons of orf24 and orf25 are part of the TrbR regulon. These results were validated using qRT-PCR experiments. It was also identified that orf16 and orf17 are not part of the TrbR regulon. The absence of conjugative plasmid pRleVF39a in donors negatively affected plasmid transfer but this result was contradicted by qRT-PCR experiments that showed higher levels of mRNA transcripts for orf23, orf24, orf26, orf27 and orf28 in a pRleVF39a cured background.Item Open Access Critically Re-evaluating Carbohydrate Metabolism in Pseudomonas aeruginosa(2019-09-19) Nguyen, Austin; Lewis, Ian A.; Storey, Douglas G.; Harrison, Joe J.; DeVinney, RebekahSeveral studies have linked carbon metabolism to a variety of different factors in P. aeruginosa. For example, the production of gluconate and 2-ketogluconate has been linked to exotoxin production, antibiotic resistance, and iron acquisition. However, glucose catabolism is known to participate in P. aeruginosa aerobic respiration. We hypothesize that, rather than factors such as iron acquisition, P. aeruginosa gluconate and 2-ketogluconate production is instead driven by the energetic needs of the cell. We developed a targeted LC-MS method and analyzed P. aeruginosa glucose metabolism production in a variety of limiting conditions. From our data, we made a model of P. aeruginosa metabolism where energy production is decoupled from biomass generation via periplasmic glucose oxidation. This model introduces a regulatory mechanism of carbon catabolism that explains the production of gluconate and 2-ketogluconate through cellular energy demands.Item Open Access Digestomics: elucidating protein catabolism through the quantitative mapping of in vivo, endogenous peptides to the proteome at an amino acid level of resolution.(2020-03-13) Bingeman, Travis Shane; Storey, Douglas G.; Lewis, Ian A.; Vogel, Hans J.; Harrison, Joe J.Plasmodium falciparum is the most lethal malaria-causing parasite. During the human stage of its lifecycle it invades erythrocytes and rapidly degrades 65-70% of the cytoplasmic hemoglobin. Despite extensive in vitro investigation of the proteases responsible for this impressive catabolic activity, the definitive purpose of this digestion remains unresolved. Many of the known proteases have been knocked out yet these parasites have minimal growth phenotypes. The common explanation is that proteolytic redundancy allows parasites to survive. I, however, hypothesize that the protease(s) essential for hemoglobin catabolism have yet to be identified. To test this, I developed a new approach to directly investigate in vivo protein catabolism. I analyze a panel of knockout parasites and show that in vivo proteolytic cleavage patterns are unchanged in knockouts, find evidence that published cut sites match poorly to in vivo data and find convincing proteolytic patterns suggesting the presence of previously unidentified cut sites.Item Open Access The effects of diketopiperazines on the virulence of Burkholderia cepacia complex species(2021-01-15) Jervis, Nicole Marie; Storey, Douglas G.; Harrison, Joe J.; Parkins, Michael D.; Savchenko, Alexei; Storey, Douglas G.Recognized as a novel class of quorum sensing inhibitors (QSIs), 2,5-diketopiperazines (DKPs) are small, organic molecules that hold many important physiochemical properties which has led to recent inquiries into their effects towards limiting the pathogenicity of multi-drug resistant bacterial pathogens. Species within the Burkholderia cepacia complex (Bcc) are one such group of pathogens that require attention towards the development of alternative therapeutic strategies given their detrimental clinical outcomes, particularly in patients with cystic fibrosis. By targeting Bcc QS regulation, our data demonstrated DKPs, cyclo(-D-ala-val), cyclo(-pro-val), cyclo(-leu-pro), and cyclo(-phe-pro) to alter the production of extracellular virulence factors and to decrease the production of biofilm-associated factors which comprise the protective extracellular matrix of Bcc biofilms. Further analysis demonstrated these DKPs to also possess antibiotic potentiator activity, enhancing the antimicrobial activity of ceftazidime, meropenem, and tobramycin. Taken together, the data collected in this study offers an initial step towards understanding the potential role of DKPs in the development of alternative therapeutics.Item Open Access A heat-activated protein expression system for biotechnology(2019-12) Fournier, Nicolas; Harrison, Joe J.; Chua, Gordon; Facchini, Peter J.The thermosensory diguanylate cyclase (TdcA) enzyme found in the Pseudomonas aeruginosa strain CF39S produces cyclic diguanylate (c-di-GMP) in response to increases in temperature. The application of TdcA in a heat-activated protein expression system was explored as an alternative to conventional induction systems. C-di-GMP-dependent gene expression measurements were conducted to investigate gene expression levels in Pseudomonas species in the presence of TdcA. An E. coli K12 mutant library was also used to investigate temperature-dependent gene expression levels for the development of a heat-activated protein expression system. Results demonstrated the thermostatted activity of TdcA in Pseudomonas species, while multiple molecular determinants were found to contribute to temperature-dependent biofilm formation and c-di-GMP-dependent gene expression levels in E. coli K12 species. Of these determinants, the c-di-GMP specific phosphodiesterase PdeO was identified as a significant contributor. Further investigation into the applicability of heat-activated expression systems would be of great benefit to the biotechnology sector.Item Open Access Impact of municipal wastewater effluent exposure on the transcriptome of fathead minnows(2019-09-19) Hooey, Christopher James; Vijayan, Mathilakath M.; Harrison, Joe J.; Cobb, John A.Municipal wastewater effluent (MWWE) is a mixture of nutrients and contaminants, including pharmaceuticals and personal care products (PPCPs), released into our waterways affecting non-target animals such as fishes. The objective of this thesis was to examine the impact of MWWE exposure on a native fish species in the Bow River. The hypothesis was that MWWE exposure disrupts molecular responses, potentially altering organismal physiology and fitness in the fathead minnow (FHM: Pimephales promales). Caging of FHM at the site closest to the wastewater treatment plant (WWTP) in the Bow River resulted in altered liver transcripts of genes involved in xenobiotic metabolism, cholesterol metabolism, and oxidative stress. Also, the livers of fish caged at the site near the WWTP showed altered methylation patterns compared to the upstream site suggesting epigenome modifications in response to MWWE exposure. Offspring of the MWWE exposed fish bred and reared in clean water also showed modulation of gene expression in the liver, similar to that of their parents, suggesting heritable effects due to parental exposure to MWWE. Exposure of FHM larvae to MWWE at dilutions similar to that reported in the Bow River disrupted the transcript abundance of genes involved in metabolism and oxidative stress, and this corresponded with diminished cortisol stress performance and altered larval behaviour. Overall, MWWE exposure disrupts the molecular responses essential for stress, growth and xenobiotic metabolism in the FHM, and some of these changes are evident even in the next generation. The results indicate that MWWE exposure affects genome methylation patterns, and suggest that epigenome modification may be playing a role in the multigenerational effects seen due to parental exposure in the FHM.Item Open Access Interactions between Serum Albumin Proteins and Polystyrene Nanoparticles(2018-07-10) Bishop, Amanda Iris; Cramb, David Thomas; Heyne, Belinda J. M.; Ling, Chang-Chun; Harrison, Joe J.Nanoparticles (NPs) have become increasingly popular for several applications, especially regarding biomedical applications because of their unique properties. However, when a nanoparticle enters a biological medium, it is thought to become encapsulated in proteins and other biomolecules in a coating termed a “protein corona.” This coating is significant as it can change the identity and surface properties of the nanoparticle, thus affecting its fate within the biological medium. Studies on the formation of NP-protein complexes have been ongoing for years although the interactions are still not fully understood due to their dynamics and complexity. As a result, this hinders the use of NPs to their full potential in biomedical applications. The studies performed in this thesis analyze the interactions of fluorescent polystyrene nanoparticles (FS) of two different sizes with bovine serum albumin proteins (BSA) by the technique of Two-Photon Excitation Cross-correlation Spectroscopy (TPE-FCCS). These interactions were explored both thermodynamically and kinetically to gain insight into the formation of the early hard corona and the kinetics of the formation of the BSA-FS complexes. The results suggested very low binding ratios and a mechanism of protein association dependent on the size of the sphere present in solution. The results also suggested an irreversible formation of BSA-FS complexes, in which the BSA appears to stack at the surface of the FS. These findings are significant as they challenge the current beliefs on the formation of a protein corona and perceived monolayer formation, and furthermore, provides a deeper understanding of NP-protein interactions.Item Open Access Intrinsic silver resistance of Pseudomonas aeruginosa in simulated human wound fluid(2020-10-19) Ostaszewski, Alexandra; Harrison, Joe J.; Schryvers, Anthony Bernard; Storey, Douglas G.; Turner, Raymond JosephA meta-analysis of randomized clinical trials using silver suggests that it does not improve patient outcomes and that the opportunistic pathogen Pseudomonas aeruginosa can persist in wounds treated with silver-impregnated bandages. However, little is known about how this microorganism withstands silver toxicity. Using strand-specific RNA-sequencing of burn-wound isolated P. aeruginosa in tandem with susceptibility testing of transposon mutants in simulated human wound fluid, we identified 46 differentially expressed genes that are bona fide silver resistance determinants. We focused on the most highly differentially expressed gene identified in the screen, copZ2. This gene is a paralog of P. aeruginosa copZ1, which encodes a copper-binding chaperone protein. CopZ2 is a small protein (65 amino acids, 6.9 kDa) that contains a M-X-C-X-H-C metal-binding motif that is essential for its function. Although CopZ1 is a putative cytoplasmic protein regulated by the copper-responsive regulator, CueR, we provide evidence that CopZ2 is enriched in the outer membrane of P. aeruginosa and that its transcription is regulated by both the putative envelope stress-response regulator, CpxR, and CueR. Purified CopZ2 binds to silver (Ag+) with an equilibrium dissociation constant (Kd) of 7.7 µM, providing it with higher affinity for silver ions than copper ions (Cu2+, Kd = 37.0 µM) in the same milieu. CopZ2 orthologues are widely distributed in Pseudomonas species, and heterologous expression of these genes confers silver tolerance to Escherichia coli. Taken together, these data begin to define the determinants of P. aeruginosa intrinsic silver resistance in environments with complex organics, and identify that CopZ2 is an evolutionarily conserved, membrane-associated, silver-binding protein that may account for a widespread ability of Pseudomonads to withstand silver toxicity.Item Open Access Investigating the role of the cell envelope in E. coli relating to silver sensitivity and resistance(2018-05-11) Westersund, Connor; Turner, Raymond Joseph; Gedamu, Lashitew; Harrison, Joe J.Silver (I) is an antimicrobial agent that has established antimicrobial activity, yet the mechanism of action is unclear. This thesis follows up on an observation that Ag+ ions cause the cell membrane to separate from the cell wall in Escherichia coli. Data from a chemical genomic screen was utilized, identifying 6 Ag-responsive cells with single gene deletions (damX, rodZ, minC, sanA, ybhO, tolB), which are genes involved in maintenance of the cell envelope. When these mutants were grown in the presence of Ag+, cells demonstrated extensive cell envelope damage as seen by transmission electron microscopy. Evaluation of kill curves, some mutants conferred resistance and others sensitivity to silver in comparison to wildtype. From the data collected, it was recognized that functions around cell wall and transport across the cell membranes have roles in both silver resistance and sensitivity.Item Open Access Investigation of the Co-occurrence of Zinc and Copper Resistance and Antimicrobial Resistance in Escherichia coli from Beef Cattle Production Systems(2020-09-21) Guardado Servellón, Fernando Joaquín; Liljebjelke, Karen A.; Checkley, Sylvia L.; Turner, Raymond Joseph; Harrison, Joe J.The present set of studies was directed towards determining the relationship between zinc (Zn) and copper (Cu) resistance and antimicrobial resistance in Escherichia coli from beef cattle production systems. First, a spectrophotometric assay was developed to properly assess the resistance levels of both Zn and Cu in Escherichia coli. The method was standardized for E. coli and it displayed a good linear dynamic range (R2>0.95), and precision (RSD< 35% in all but three Zn concentrations). The MIC for the E. coli reference strain (ATCC 25922) were 2.78 mol/ml and 8.41 mol/ml for Zn and Cu respectively. In the second study, we determined antimicrobial phenotypes and the Zn and Cu resistance levels for E. coli isolates from environmental samples obtained from W.A Ranches. The samples analyzed had a low prevalence of antimicrobial resistance as 31/39 isolates were susceptible to all the antimicrobials tested using the disc diffusion method. The most common resistance was ampicillin (4/39) and amoxicillin-clavulanic acid (4/39), with one isolate being resistant to doxycycline. All the isolates resistant to ampicillin were also resistant to amoxicillin-clavulanic acid. The isolates were then tested for Zn and Cu resistance using the assay developed. Seven of the eight isolates with resistant or intermediate antimicrobial resistance patterns showed higher optical density (OD) in the Zn and Cu resistance spectrophotometric assay when compared to the reference strain. Fisher’s exact test was conducted to compare antimicrobial-resistant (all antimicrobials) and susceptible isolates in their resistance to Zn and Cu, with the results showing that the antimicrobial-resistant isolates are also more likely to have a higher resistance to Zn (p value<0.001) and Cu (p value=0.013) as indicated by higher absorbance units (AU). A Principal Component Analysis showed the clustering of 6/8 antimicrobial-resistant isolates based on the Zn and Cu resistance meaning that resistance to these metals might be an indicator of antimicrobial resistance.Item Open Access Multimetal resistance and tolerance in microbial biofilms(2008) Harrison, Joe J.; Turner, Raymond J.; Ceri, HowardItem Open Access NK cell killing of extracellular Pseudomonas aeruginosa(2020-06-26) Feehan, David; Mody, Christopher Hugh; Cobo, Eduardo R.; Harrison, Joe J.Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects individuals with the respiratory illness, Cystic Fibrosis and contributes to airway blockage and loss of lung function. NK cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes. The hypothesis of this project is that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co¬-culture of YT cells, a human NK cell line, and GFP P. aeruginosa stained with the membrane viability dye propidium iodide, demonstrated that YT cell killing of P. aeruginosa is contact mediated. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa, as determined by CFU count. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of PAO1 in comparison to wild type controls. This demonstrates that granzymes are required for killing, but exhibit redundancy in their function. In summary, these results suggest that NK cell kill P. aeruginosa through a membrane damaging, contact-dependent process, requiring granzymes.