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.

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.

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.

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.

Mycobacterium abscessus (Mabs) is a multidrug-resistant nontuberculous mycobacterium capable of causing persistent pulmonary infection. It most prominently threatens those with cystic fibrosis (CF), a progressive and genetic disorder characterized by an immunocompromised respiratory tract. Current treatments fail to eradicate Mabs, meaning novel alternatives are greatly needed. Antimicrobial peptides (AMPs) are short sequences of amino acids that display broad-spectrum antimicrobial activity and play an important role in innate immunity. To maximize their therapeutic potential, key AMP features can be rationally combined through an iterative engineering process to create synthetic, designed AMPs (dAMPs). In this investigation, two dAMPs, RP554 and RP557, reduced Mabs ATCC 19977 viability by 99.99% and were subjected to further testing. In antimicrobial susceptibility testing with Mabs ATCC 19977, RP554 and RP557 demonstrated bactericidal activity at concentrations 16-32 μM. Complete killing of Mabs ATCC 19977 by RP554 and RP557 occurred rapidly in <24 h. RP554 and RP557 also inhibited 20 Mabs clinical isolates obtained from CF patients. Furthermore, RP554 and RP557 retained anti-Mabs activity after pre-exposure to human serum, indicating potential stability in blood. Conversely, the tested dAMPs did not kill Mabs during in vitro experiments in an artificial sputum medium. Novel antimicrobials, such as the RP554 and RP557 dAMPs, offer therapeutic potential for otherwise resistant bacterial pathogens, including Mabs, that afflict both CF and non-CF patients.

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.

Coccidioidomycosis or Valley Fever (VF) is an emerging fungal respiratory infection endemic to the southwest region of the United States, and parts of Mexico, Central and South America. Satellite cases have also been reported in Washington and Oregon. It is estimated that in Maricopa County alone, VF accounts for 10-30% of community-acquired pneumonia. Difficulty in diagnosis is largely attributed to lack of antibody reactivity to antigens used in diagnosis, especially early in disease. Serological detection of VF employs mycelial-phase culture filtrates as antigen. While culture filtrates are thought to provide the most specific diagnostic antigen, preparation includes the growth of large volume Coccidioides cultures which require employment of extensive safety precautions in a BSL3 setting. An additional concern with use of culture filtrates as an antigen source is batch variability, as expression of immunogenic proteins within each lot are variable. To address safety and batch variability concerns, this thesis proposes the use of recombinant Coccidioides proteins as a consistent and reliable antigen source. For the purpose of this study, I expressed known antigenic Coccidioides proteins in a eukaryotic, recombinant protein expression system. Recombinant endochitinase-1 (rCTS1) and recombinant heat-labile antigen (rHL-Ag) were evaluated for serologic reactivity by ELISA, using a sample set of 55 known serologically positive and 55 known negative human sera specimens, previously tested in Mayo Clinic Arizona (MCA) serologic laboratories. Evaluation by ELISA demonstrated 94.55% sensitivity and 92.72% specificity using combined rCTS1 and rHL-Ag as an antigen source, indicating promising diagnostic utility.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen commonly associated with increased morbidity and mortality in cystic fibrosis (CF) patients. To adapt to the CF lung environment, P. aeruginosa undergoes multiple genetic changes as it moves from an acute to a chronic infection. The resultant phenotypes have been associated with chronic infection and can provide important information to track the patient’s individualized disease progression. This study examines the link between the accumulation of QS genetic mutations and phenotypic expression in P. aeruginosa laboratory strains and clinical isolates. We utilized several plate-based and colorimetric assays to quantify the production of pyocyanin, rhamnolipids, and protease from paired clinical early- and late-stage chronic infection isolates across 16 patients. Exoproduct production of each isolate was compared to the mean production of pooled isolates to classify high producing (QS-sufficient) and low producing (QS-deficient) isolates. We found that over time P. aeruginosa isolates exhibit a reduction in QS-related phenotypes during chronic infections. Future research of the QS regulatory networks will identify whether reversion of genotype will result in corresponding phenotypic changes in QS-deficient chronic infection isolates.

Persons with cystic fibrosis (CF) are highly susceptible to lung infections caused by the opportunistic pathogens Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA). By age 20, ~16% of CF patients have co-infections with these two bacteria, and this number grows as the patients age1. PA-SA co-infections are associated with worsened clinical outcomes in CF patients, but the reasons are not well understood. One hypothesis is that SA influences the production of PA virulence factors and other chronic infection phenotypes. Previous work in our lab investigated the effects of SA on PA quorum-regulated phenotypes when they are grown as planktonic co-cultures. We are expanding on this result by testing whether SA can influence PA phenotypes without being in direct contact, and without being able to exchange soluble secreted factors. In this study, we hypothesized that SA produces volatile organic compounds (VOCs) that cause changes in PA phenotypes leading to a down-regulation of motility and protease production, and increased antibiotic resistance. To test this hypothesis, we exposed two laboratory strains of PA to the VOCs produced by pre-grown lawns of two strains of SA, and measured PA motility by conducting swarming, swimming, and twitching assays, measuring protease production, as well as antibiotic sensitivity. After exposing PA to a pre-grown lawn of SA, there was a significant difference in some phenotypes compared to controls. There were significant decreases in swarming motility, twitching motility, and protease production, and an increase in a bright green pigment (possibly siderophores) when PA was exposed to SA. The degree of phenotypic alterations was dependent on both the PA strain and the SA strain being tested. Exposure to SA VOCs also altered PA sensitivity to ciprofloxacin, though one strain caused an increase in susceptibility while the other SA strain caused an increase in resistance. These data demonstrate that SA VOCs can influence PA phenotypes in vitro, which may have relevance for CF patients who are co-infected with these two bacteria.

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.