Theses and Dissertations at Montana State University (MSU)
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Item Bacterial cultivation in microscale drops and capsules to resolve single-cell growth physiology(Montana State University - Bozeman, College of Engineering, 2023) Pratt, Shawna Leigh; Chairperson, Graduate Committee: Ross Carlson; This is a manuscript style paper that includes co-authored chapters.Single-cell heterogeneity contributes to the complex population dynamics of infectious microbial communities. Improving our understanding of single-cell physiology and heterogeneity may aid in mitigating microbial infections; however, assaying large populations of single cells can be challenging. Despite recent developments in single-cell assaying, tracking the physiology of large numbers of individual cells and their lineages over time is difficult to achieve using current technologies. Here, I apply drop-based microfluidics to develop microscale tools for improving high-throughput single-cell microbial growth assays. Drop-based microfluidics is a technology that generates and manipulates microscale drops. In this work, I create water-in-oil drops and hydrogel-shelled microcapsules using drop-based microfluidics to study the growth of P. aeruginosa bacteria, a key pathogen implicated in chronic lung infections and wounds. The growth of single bacterial cells inside drop microcompartments is observed via time-lapse confocal microscopy. Bacteria were cultured in water-in-oil drops and prepared for long-term storage in a novel microfluidic device environment, which we call a DropSOAC (Drop Stabilization on a Chip) chamber. The DropSOAC method prevents drop destabilization by saturating microfluidic devices with equilibrated water and oil, maintaining phase equilibrium in the drop emulsion. Using DropSOAC, the single-cell growth of starved P. aeruginosa wildtype and hibernation promotion factor mutants were characterized, revealing significant growth heterogeneity in the mutant strain. Finally, we present a method for generating hydrogel-shelled microcapsules that enables the culturing of single cells in microscale environments where nutrients and waste can diffuse in and out of the microculture environment. A 3-D microfluidic device and capsule generation protocol are designed, resulting in an optimized approach for capsule production using phase-separating polymer systems and rapid hydrogel crosslinking. The growth of hundreds of individual P. aeruginosa cells is observed over time with the hydrogel- shelled microcapsules. Due to the permeability of the microcapsules, antibiotics can be introduced at various times during growth to investigate single and biofilm P. aeruginosa physiology. Overall, this work introduces novel approaches for high-throughput, single-cell microbial growth characterization that enables a deeper understanding of the role of heterogeneity in bacterial populations.Item Structure and function of a prokaryotic argonaute from Pseudomonas aeruginosa(Montana State University - Bozeman, College of Agriculture, 2020) Erickson, Reece Sheridan; Chairperson, Graduate Committee: Blake WiedenheftArgonautes (Ago) are structurally and functionally diverse proteins present in all domains of life. A common feature of these ancient proteins is their ability to bind nucleic acid guides that target the protein to complementary sequences. Although eukaryotic argonautes (eAgo) have been well-studied, we still know very little about the function of prokaryotic argonautes (pAgo) in bacterial and archaeal species. To address this gap in our knowledge, my thesis focused on determining the biochemical properties as well as the cellular functions of a pAgo from the organism Pseudomonas aeruginosa PACS2 (PaAgo). Here, we show that PaAgo plays a role in regulating the expression of transposons within PACS2. I also present results indicating that deletion of the PaAgo gene and its neighboring genes causes toxicity to P. aeruginosa. Finally, I provide evidence that PaAgo and a neighboring protein are binding partners and form a multi-protein complex. Future work will focus on copurifying and sequencing PaAgo nucleic acid guides as well as clarifying the mechanisms guide acquisition and biological function.Item Quantitative 1 H NMR analyses of immunometabolic modulation in human macrophages(Montana State University - Bozeman, College of Letters & Science, 2019) Fuchs, Amanda Lee; Chairperson, Graduate Committee: Valerie Copie; Sage M. Schiller was an author and Wyatt J. Keegan, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Quantitative 1 H NMR metabolomics reveals distinct metabolic adaptations in human macrophages following differential activation' in the journal 'Metabolites' which is contained within this dissertation.; Sage M. Schiller was an author and Isaac R. Miller, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Pseudomonas aeruginosa planktonic- and biofilm-conditioned media elicit divergent responses in human macrophages' submitted to the journal 'PLoS pathogens' which is contained within this dissertation.Macrophages are innate immune cells that are found ubiquitously in nearly all human tissues, where they support host innate and adaptive immune responses in an effort to maintain systemic homeostasis. They are inherently plastic in nature and can dramatically modulate their functional phenotype according to pathogen and microenvironmental stimuli. Previous studies have shown that macrophages are particularly important for the resolution of inflammation in acute wound healing, which is marked by a phenotypic transition of wound macrophages from pro-inflammatory to anti-inflammatory. Chronic, or non-healing, wounds, such as diabetic, pressure, and venous leg ulcers, feature a prolonged host inflammatory response due in part to aberrant wound macrophage behavior. Non-healing in chronic wounds has also been shown to be dependent upon the establishment of pathogenic biofilms, which are more resistant to host defense mechanisms than planktonic, or free-floating, bacteria. Therefore, investigating macrophage dysregulation in the presence of bacterial biofilms has gained considerable interest. Here, 1D 1 H NMR-based metabolomics was utilized to identify metabolic pathways that are differentially modulated following primary human monocyte-derived macrophage activation with pro-inflammatory or anti-inflammatory stimuli relative to resting macrophages. Metabolic profiling of inflammatory macrophages indicated a substantial increase in oxidative stress as well as a decrease in mitochondrial respiration. These metabolic profiles also provided evidence that inflammatory macrophages divert metabolites from de novo glycerophospholipid synthesis to inhibit oxidative phosphorylation. In addition, we investigated which metabolic pathways are differentially modulated following primary human monocyte-derived macrophage exposure to Pseudomonas aeruginosa planktonic- and biofilm-conditioned media. Metabolic profiling of PCM- and BCM-exposed macrophages indicated a significant depletion of intracellular glucose without elevation of downstream glycolytic products. These metabolic patterns suggest that PCM- and BCM-exposed macrophages potentially divert glycolytic intermediates towards inositol phosphate metabolism. Overall, our studies provide additional support to previous findings, generate novel results regarding metabolic modulation of human macrophages following activation and exposure to planktonic- vs. biofilm-conditioned media, and contribute new insight to the field of immunometabolism.Item The role of ribosome hibernation factor and the stringent response in the survival of Pseudomonas aeruginosa during dormancy(Montana State University - Bozeman, College of Letters & Science, 2019) Theng, Sokuntheary; Chairperson, Graduate Committee: Michael FranklinPseudomonas aeruginosa survives in a dormant state in low nutrient environments and is able to resuscitate when favorable conditions are available. In response to stressful environmental conditions including antibiotic stress, osmotic stress, and starvation, P. aeruginosa undergoes the (p)ppGpp-mediated stringent response to induce a variety of genes for entry into the dormant state. One critical mechanism for P. aeruginosa dormancy involves in inactivating translation machinery by converting active ribosomes into inactive 70S and 100S ribosome monomers and dimers. Hibernation promoting factor (HPF~11.6kDa) is a ribosome-associated protein that stabilizes inactive ribosomes. Here, I investigated the relationship between HPF and the stringent response in survival of P. aeruginosa during dormancy. I also investigated role of HPF preserving ribosomes in dormant cells by quantifying the abundance of two ribosomal proteins (L5 of the large ribosomal subunit and S13 of the small ribosomal subunit), during P. aeruginosa starvation. For quantitative analysis during nutrient-limited conditions, I used immunoblotting and image analysis to quantify L5 and S13 abundances in the wild-type strain, PAO1, and in a relA/spoT double mutant strain, which is incapable of producing (p)ppGpp. The results show that the relA/spoT mutant loses HPF proteins after four days of starvation. To explore the role of HPF in preserving ribosomal proteins, I quantified L5 and S13 in wild-type PAO1 and in the Delta hpf deletion mutant and in the relA/spoT mutant. Immunoblots showed that both L5 and S13 rapidly decrease by day 2 of starvation in the Delta hpf mutant strain, but that these proteins are maintained throughout eight days of starvation in the wild-type strain. Notably, L5 and S13 are maintained in the Delta relA/Delta spoT strain throughout starvation. Lastly, I determined if the amount of cellular HPF required for P. aeruginosa ribosome maintenance. Growth in minimal medium (MOPS medium) affects the amount of HPF produced, based on the carbon source. Therefore, I tested ribosome maintenance during starvation of cells first cultured in MOPS-fructose or MOPS-glucose. The results indicate that HPF production during growth in MOPS-fructose is higher than in MOPS-glucose. However, the increased amount of HPF did not affect the amount of L5 and S13 during starvation. Therefore, the amount of HPF is not critical for P. aeruginosa to maintain its ribosomes during starvation. These results demonstrate HPF is essential for maintenance of ribosomal proteins during starvation of P. aeruginosa, and that the ribosomal proteins are likely degraded in the absence of HPF. P. aeruginosa needs a minimum amount of HPF to preserve ribosomes during nutrient-limited condition.Item Characterization of the stability of Pseudomonas aeruginosa ribosomal proteins under stress conditions(Montana State University - Bozeman, College of Letters & Science, 2017) Yanardag, Sila; Chairperson, Graduate Committee: Michael FranklinIn this study, I aimed to standardize western blot methods for probing large and small ribosomal subunits of Pseudomonas aeruginsa grown under different environmetal conditions, and to characterize the stability of ribosomal proteins to bring light to the heterogeneous composition of the population, which is hypothesized as one mechanism for antibiotic tolerance. Long-term studies done with P.aeruginosa PAO1 showed that mRNA transcripts of two proteins, RMF and HPF, are highly abundant at the biofilm-nutrient interface of the thick P.aeruginosa biofilms. Also, it was previously shown by Perez et al. and Williamson et al. (Pérez-Osorio et al., 2010; Williamson et al., 2012) that the cells located at the oxygen limited interphase of the biofilm were metabolically inactive or slow-growing. Akiyama et al. (Akiyama et al., 2017) and Williamson et al. (Williamson et al., 2012) found that HPF is a critical protein for the maintenance of 23S rRNA and overall ribosomal RNA stability after prolonged stress exposure (Akiyama et al., 2017; Williamson et al., 2012). In light of this information, Akiyama et al. (Akiyama et al., 2017) showed that in the absence of the HPF protein, P.aeruginosa cannot protect its ribosome integrity and cannot resuscitate from dormancy after the environmental stressors are gone. Perez et al. (Pérez-Osorio et al., 2010) showed that P.aeruginosa biofilms are heterogeneous in physiology, and it is posited that persister cells of the biofilm are located at the bottom of the biofilm, unaffected by the antibiotic exposure and therefore can repopulate the biofilm (Williamson et al., 2012). Localization of ribosomal subunits and determination of the abundance of ribosomes within the heterogeneous biofilms will provide valuable insights on the mechanisms of persister cell formation, dormancy, and resusication from dormancy. In order to do so, I have isolated two ribosomal proteins, L5 and S13, and HPF. In this study, I generated polyclonal antibodies against those three proteins. I used the antibodies to determine the abundance of these proteins during the normal course of growth of the wild type and Deltahpf mutant strains. Growth analysis in nutrient rich media gave us an understanding of the stability of 70S ribosomes when the bacterium was growing without any stress. Later, the wild type and Deltahpf strain were grown in a carbon and nitrogen-limited environment for seven days to examine the response of the cells to the starvation stress regarding ribosomal stability. Finally, I tested the hypothesis that cells located at the bottom of the biofilm are abundant in the HPF protein, and therefore contain more inactive ribosomes compared to the cells located at the top of biofilm.Item Investigation of a control strategy for manipulation and prevention of Pseudomonas aeruginosa PAO1 biofilms in metalworking fluids(Montana State University - Bozeman, College of Engineering, 2018) Ozcan, Safiye Selen; Chairperson, Graduate Committee: Christine Foreman; Markus Dieser, Albert E. Parker, Narayanaganesh Balasubramanian and Christine M. Foreman were co-authors of the article, 'Quorum sensing inhibition as a promissing method to control biofilm growth in metalworking fluids' submitted to the journal 'Environmental science & technology' which is contained within this thesis.Microbial contamination in metalworking fluid (MWF) circulation systems is a serious problem. Particularly water based MWFs promote microbial colonization despite the use of biocides. Inhibiting the quorum sensing mechanism (i.e. cell-cell communication) in bacteria is a promising approach to control and prevent biofilm formation. The objective of this study was (i) to determine the microbial community in MWFs from operational machining shops, (ii) to investigate the effect of well-known quorum sensing inhibitors on controlling biofilm formation, and (iii) to implement experimental data from selected enzymes to a computer simulation biofilm accumulation model (BAM). Planktonic and biofilm samples from two local machining shops in Bozeman, MT, were collected to determine the extent of microbial colonization. In both operations, microbial communities were dominated by Pseudomonadales (60.2-99.7%). Rapid recolonization was observed even after dumping spent MWFs and cleaning. Considering the dominance of Pseudomonadales in MWFs, the model organism Pseudomonas aeruginosa PAO1 was selected for testing the effects of quorum sensing inhibitor compounds on biofilm formation. From a variety of enzymes, natural, and chemical compounds screened for quorum sensing inhibition, Patulin (40microns) and Furanone C-30 (75microns), were found to be effective in reducing biofilm formation in MWFs when applied as single compound amendments and in combination with the polysaccharide degrading enzyme alpha-amylase from Bacillus amyloliquefaciens. Particularly Furanone C-30, as a single amendment and in combination with alpha-amylase decreased biofilm formation by 76% and 82% after 48 hours. Putatively identified homoserine lactones in MWFs treated with Furanone C-30 provided evidence for quorum sensing inhibition on biofilm formation. BAM was employed to study the effect of alpha-amylase (3 Units mL -1) on P. aeruginosa PAO1 biofilms in batch reactors for 24 and 48 hours. In the absence of alpha-amylase, biofilm thickness was predicted to be 23.11 and 31.37 microns, while its presence reduced thickness to 10.47 and 13.07 microns after 24 and 48 hours, respectively. The results presented herein highlight the potential effectiveness of quorum sensing inhibition as a strategy to reduce biofilms in MWFs.Item The roles of hibernation promoting factor in resuscitation of Pseudomonas aeruginosa from dormancy(Montana State University - Bozeman, College of Letters & Science, 2018) Akiyama, Tatsuya; Chairperson, Graduate Committee: Michael Franklin; Kerry S. Williamson, Robert Schaefer, Shawna Pratt, Connie B. Chang and Michael J. Franklin were co-authors of the article, 'Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation' in the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this thesis.; Kerry S. Williamson and Michael J. Franklin were co-authors of the article, 'Expression and regulation of the Pseudomonas aeruginosa hibernation promoting factor' submitted to the journal 'Molecular microbiology' which is contained within this thesis.Microbial biofilms are surface-attached communities of microorganisms. Biofilms are often associated with chronic infections due to antibiotic resistance. Pseudomonas aeruginosa causes chronic pulmonary infections in cystic fibrosis patients and chronic wound infections in diabetic ulcers. One mechanism for biofilm-associated resistance is a formation of non-dividing, metabolically dormant cells resisting antibiotics. The goals of this research were to understand the molecular mechanisms involved in formation, maintenance, and resuscitation of dormant cells, with the ultimate goal of developing enhanced treatment strategies for chronic biofilm-associated infections. While dormant, bacteria must maintain cellular and macromolecular integrity required for resuscitation. Previous study found the high abundance of messenger RNAs for ribosome accessory proteins, hibernation promoting factor (HPF) and ribosome modulation factor (RMF), in the dormant subpopulation of P. aeruginosa biofilms. In this research, we characterized the activity and expression of the ribosome hibernation factor. By exposing the hpf and rmf deletion mutant strains to nutrient starvation, we found that HPF, but not RMF, is essential for cell viability maintenance during starvation-induced dormancy. Viability loss in the hpf mutant strain corresponded to loss of ribosomal RNA, and by inference, loss of cellular ribosome content during dormancy. Single-cell level studies using fluorescence in situ hybridization showed the heterogeneous ribosomal RNA levels for both the hpf and wild-type cells. Single-cell level studies using drop-based microfluidics also showed heterogeneity in resuscitation from dormancy. While the majority lost ability to resuscitate from dormancy, a fraction of hpf mutant cells recovered but with an extended lag time. We also determined the regulation of HPF expression using a transposon-based yellow fluorescent protein (YFP) reporter fused to HPF. The results showed that hpf is expressed from at least two different promoters. HPF expression is also controlled by mRNA folding, and an autofeedback mechanism. The complex regulatory mechanism at transcriptional and post-transcriptional levels may allow the bacteria to respond to nutrient limitation and enter a dormant state. Our results show the importance of HPF on ribosome preservation during starvation, as well as how this hibernation factor is regulated. The results provide new information of this novel target for treatment of dormant infectious bacteria.Item Understanding the physiology of Pseudomonas aeruginosa biofilms in an in vitro chronic wound model(Montana State University - Bozeman, College of Letters & Science, 2016) White, Benjamin Michael; Chairperson, Graduate Committee: Michael FranklinPseudomonas aeruginosa is a common colonizer of cutaneous abrasions and burns. These Gram-negative, aerobic bacteria are problematic due to their natural resilience to antibiotics and their metabolic versatility. P. aeruginosa can produce a prodigious extracellular matrix. Within this matrix P. aeruginosa can divide and form a multicellular community called a biofilm. Biofilms have become a health concern worldwide, as these communities are highly resistant to antibiotics. This thesis reports the effort to model the wound environment. A chronic wound exudate medium was designed and P. aeruginosa was grown at 33°C under low flow in a drip-flow biofilm reactor. Bacterial cells were grown planktonically and in biofilms. Biofilms were treated with the fluoroquinone, ciprofloxacin for 24 hours and transcriptomic and metabolomic data were collected from treated and untreated biofilms and planktonic cells. Cells growing in biofilms demonstrated a shift in in the regulation of their tricarboxylic acid cycle, amino acid degradation, and siderophore biosynthesis genes as compared to planktonic cells. Ciprofloxacin treatment altered the transcriptomic landscape within the biofilm. Changes were observed in the transcription of DNA repair, prophage, and phenazine biosynthesis genes. An important virulence factor, the type VI secretion system, was also differently regulated in these samples and is likely important for the persistent infection of wounds. From the information collected, target genes have been identified for future gene-knockout and ciprofloxacin susceptibility assays. A reduction in fitness may indicate genes that are relevant drug targets to enhance antibiotic treatment of these resilient communities.Item The role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide(Montana State University - Bozeman, College of Agriculture, 1999) Elkins, James GarrettItem Interaction between human neutrophils and Pseudomonas aeruginosa biofilm : morphological and biochemical characterization(Montana State University - Bozeman, College of Agriculture, 2003) Papke, Maiko Sasaki; Chairperson, Graduate Committee: Michael Franklin