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- Genre: Doctoral Dissertation
Description
Mycobacterial infections, as represented by leprosy and tuberculosis, have persisted as human pathogens for millennia. Their environmental counterparts, nontuberculous mycobacteria (NTM), are commodious infectious agents endowed with extensive innate and acquired antimicrobial resistance. The current drug development process selects for antibiotics with high specificity for definitive targets within bacterial metabolic and replication pathways. Because these compounds demonstrate limited efficacy against mycobacteria, novel antimycobacterial agents with unconventional mechanisms of action were identified. Two highly resistant NTMs, Mycobacterium abscessus (Mabs) a rapid-growing respiratory, skin, and soft tissue pathogen, and Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, were selected as targets. Compounds that indicated antimicrobial activity against other highly resistant pathogens were selected for initial screening. Antimicrobial peptides (AMPs) have demonstrated activity against a variety of bacterial pathogens, including mycobacterial species. Designed antimicrobial peptides (dAMPs), rationally-designed and synthetic contingents, combine iterative features of natural AMPs to achieve superior antimicrobial activity in resistant pathogens. Initial screening identified two dAMPs, RP554 and RP557, with bactericidal activity against Mabs. Clay-associated ions have previously demonstrated bactericidal activity against MU. Synthetic and customizable aluminosilicates have also demonstrated adsorption of bacterial cells and toxins. On this basis, two aluminosilicate materials, geopolymers (GP) and ion-exchange nanozeolites (IE-nZeos), were screened for antimicrobial activity against MU and its fast-growing relative, Mycobacterium marinum (Mmar). GPs demonstrated adsorption of MU cells and mycolactone, a secreted, lipophilic toxin, whereas Cu-nZeos and Ag-nZeos demonstrated antibacterial activity against MU and Mmar. Cumulatively, these results indicate that an integrative drug selection process may yield a new generation of antimycobacterial agents.
ContributorsDermody, Roslyn June (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022
Description
Mycobacterium tuberculosis (Mtb), the etiological agent of the tuberculosis disease, is estimated to infect one-fourth of the human population and is responsible for 1.5 million deaths annually. The increased emergence of bacterial resistance to clinical interventions highlights the lack in development of novel antimicrobial therapeutics. Prototypical bacterial two-component systems (TCS) allow for sensing of extracellular stimuli and relay thereof to create a transcriptional response. The prrAB TCS is essential for viability in Mtb, presenting itself as an attractive novel drug target. In Mtb, PrrAB is involved in the adaptation to the intra-macrophage environment and recent work implicates PrrAB in the dosR-dependent hypoxia adaptation. This work defines a direct molecular and regulatory connection between Mtb PrrAB and the dosR-dependent hypoxia response. Using electrophoretic mobility shift assays combined with surface plasmon resonance, the Mtb dosR gene is established as a specific target of PrrA, corroborated by fluorescence reporter assays demonstrating a regulatory relationship. Considering the scarce understanding of prrAB essentiality in nontuberculous mycobacteria and the presence of multiple prrAB orthologs in Mycobacterium smegmatis and Mycobacterium abscessus, CRISPR interference was utilized to evaluate the essentiality of PrrAB beyond Mtb. prrAB was found to be inessential for viability in M. smegmatis yet required for in vitro growth. Conversely, M. abscessus prrAB repression led to enhanced in vitro growth. Diarylthiazole-48 (DAT-48) displayed decreased selectivity against M. abscessus but demonstrated enhanced intrinsic activity upon prrAB repression in M. abscessus. Lastly, to aid in the rapid determination of mycobacterial drug susceptibility and the detection of mycobacterial heteroresistance, the large volume scattering imaging (LVSim) platform was adapted for mycobacteria. Using LVSim, Mtb drug susceptibility was detected phenotypically within 6 hours, and clinically relevant mycobacterial heteroresistance was detected phenotypically within 10 generations. The data generated in these studies provide insight into the essential role of PrrAB in Mtb and its involvement in the dosR-dependent hypoxia adaptation, advance the understanding of mycobacterial PrrAB essentiality and PrrAB-associated mycobacterial growth dependency. These studies further establish molecular and mechanistic connection between PrrAB and DAT-48 in Mtb and M. abscessus and develop a rapid phenotypic drug susceptibility testing platform for mycobacteria.
ContributorsHaller, Yannik Alex (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Plaisier, Christopher (Committee member) / Acharya, Abhinav (Committee member) / Arizona State University (Publisher)
Created2023
Description
To combat the global antimalarial resistance crisis effective resistance management strategies are needed. To do so, I need to gain a better understanding of the ecological interactions occurring within malaria infections. Despite the importance of the complex interplay among co-infecting strains, our current knowledge and empirical data of within-host diversity and malaria disease dynamics is limited. In this thesis, I explore the multifaceted dynamics of malaria infections through an ecological lens. My overall research question is: "How do ecological interactions, including niche complementarity, competition dynamics, and the cost of resistance, shape the outcomes of malaria infections, and what implications does this have on understanding and improving resistance management strategies?” In Chapter II, titled “Niche Complementarity in Malaria Infections” I demonstrate that ecological principles are observed in malarial infections by experimentally manipulating the biodiversity of rodent malaria P. chabaudi infections. I observed that some parasites experienced competitive suppression, others experienced competitive facilitation, while others were not impacted. Next, in Chapter III, titled “Determining the Differential Impact of Competition Between Genetically Distinct Plasmodium falciparum Strains” I investigate the differential effect of competition among six genetically distinct strains. The impact of competition varied between strain combinations, and both suppression and facilitation were observed, but most pairings had no competitive interactions. Lastly, in Chapter IV, titled “Assessing Fitness Costs in Malaria Parasites: A Comprehensive Review and Implications for Drug Resistance Management”, I summarize where the field currently stands and what evidence there is for the presence of a fitness cost, or lack thereof, and I highlight the current gaps in knowledge. I found that evidence from field, in vitro, and animal models are overall suggestive of the presence of a fitness cost, however, these costs were not always found. Amid the current focus on malaria eradication, it is crucial to understand the impact of biodiversity on disease severity. By incorporating an ecological approach to infectious disease systems, I can gain insights on within-host interactions and how they impact parasite fitness and transmissibility.
ContributorsSegovia, Xyonane (Author) / Huijben, Silvie (Thesis advisor) / Bean, Heather (Committee member) / Gile, Gillian (Committee member) / Hogue, Ian (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2024
Description
Peatlands are significant global carbon sinks, where plant litter accumulation outpaces the rate of microbial degradation, which can result in significant emissions of methane and carbon dioxide. The Pastaza-Marañón foreland basin (PMFB) in the western Amazon contains the largest expanse of tropical peatlands in South America, characterized by a diversity of soil properties, including pH and mineral concentration. The PMFB is predicted to decrease in its carbon capture capacity along with a rise in greenhouse gas emissions as the climate changes. Therefore, it is imperative to understand the impact that soil properties have on the abundance of functions, microbial physiology, and interspecies interactions between microbial community members. Metagenomic sequencing of soil samples from three geochemically distinct peatlands revealed site-specific enrichment of functions related to carbon, nitrogen, phosphorus, and sulfur cycling. Additionally, 519 metagenome-assembled genomes (MAGs) were recovered, revealing variations in microbial populations responsible for organic matter degradation and nutrient (nitrogen and sulfur) cycling across sites. From these MAGs, a novel family within the Bathyarchaeia was identified, Candidatus Paludivitaceae. This family is putatively capable of carboxydotrophy, able to use CO for energy and biomass. Subsequently they could detoxify the environment of CO benefiting other community members and playing an indirect role in modulating carbon cycling. To experimentally investigate interactions of peatland microbes, co-culture experiments assessed the impact of carbon substrates (4-hydroxybenzoic acid, mannitol, and arginine) on microbial interactions from heterotrophs isolated from two geochemically distinct peatlands. Results indicate substrate and peatland type significantly influence nature and frequency of microbial interactions. The response of microbial genera to carbon substrate also varied showing the role of metabolic traits and substrate preferences in determining growth patterns of microbes. This research advances our understanding of microbial ecology in tropical peatlands and better informs predictions as the climate changes.
ContributorsPavia, Michael Joseph (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Bean, Heather (Committee member) / Bouskill, Nicholas (Committee member) / Penton, Christopher (Committee member) / Arizona State University (Publisher)
Created2024
Description
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection.
ContributorsMaarsingh, Jason (Author) / Haydel, Shelley E (Thesis advisor) / Roland, Kenneth (Committee member) / Sandrin, Todd (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2019
Description
Biocrusts are microbial communities that inhabit arid soil surfaces, providing essential services to dryland ecosystems. A paradoxical filamentous cyanobacterium, Microcoleus vaginatus, resides within the biocrust. While is often pioneers the colonization of bare, nutrient-poor desert soils worldwide, it cannot fix dinitrogen. In nature, M. vaginatus coexists with a unique microbial community, a “cyanosphere”, that is characterized by a high abundance of diazotrophic heterotrophs. This suggests mutualistic relationships wherein nutrients are traded between phototrophs and heterotrophs. To explore these relationships, I performed targeted, pedigreed isolation of cyanosphere members and used co-cultivation to recreate the mutualism in culture. Results showed that, in the absence of fixed nitrogen, M. vaginatus grew well when co-cultured with cyanosphere diazotrophs, but only poorly or not at all when alone or with non-cyanosphere diazotrophs. In agreement with this, the experimental provision of nitrogen to natural populations resulted in a loss of diazotrophs from the cyanosphere compared to controls, but the addition of phosphorus did not. Additionally, the convergence of M. vaginatus trichomes into large bundles held by a common sheath was elicited in culture by the addition of cyanosphere diazotrophs, pointing to a role of cyanobacterial motility responses in the development of mutualistic interactions. I then demonstrated that the tendency of M. vaginatus to stay within bundles and close to the sheath-dwelling cyanosphere was dependent on the cyanosphere population size. This effect was likely mediated by glutamate that acted as a signaling molecule rather than as a N source and impacted the gliding speed and negative chemophobic responses on the cyanobacterium. Glutamate seems to be used as a cue to spatially optimize cyanobacterium-cyanosphere mutualistic exchanges. My findings have potential practical applications in restoration ecology, which I further pursued experimentally. Co-inoculation of soil with cyanosphere diazotrophs resulted in swifter development of biocrusts over inoculation with the cyanobacterium only. Further, their addition to disturbed native soils containing traces of cyanobacteria sufficed for the formation of cohesive biocrusts without cyanobacterial inoculation. The inclusion of such “biocrust probiotics” in biocrust restoration is recommended. Overall, this body of work elucidates the hitherto unknown role of beneficial heterotrophic bacteria in the initial formation and development of biocrusts.
ContributorsNelson, Corey (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Penton, C. Ryan (Committee member) / Gile, Gillian (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2021