Browsing by Subject "Mycology"
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Item Open Access Developing Novel Antifungal Compounds for Use as Single-Agent and in Multi-Drug Combination Therapies for Treating Invasive Fungal Infections(2023) Rivera, AngelaInvasive fungal infections are recognized as a global health threat with the World Health Organization listing 4 fungal species as critical pathogens for global study. With the rise of resistance and new pathogenic species continuing to evolve, there is an immediate need for antifungal drug development. Current antifungal therapies are antiquated, have numerous side effects, and consist of only four available classes. The development of novel drugs to treat fungal disease is hindered by the evolutionary conservation between fungi and mammals. With these considerations in mind, this thesis aims to investigate repurposing established therapeutics for use in treating invasive fungal infections.
Chapter 1 focuses on the global impact of fungal disease and highlights Cryptococcus neoformans as a primary pathogen to study. It begins by outlining the current state of fungal pathogenesis and the antifungal therapeutic armament. The development of antifungal agents will additionally be addressed by giving an overview of investigational drug candidates including FKBP12 ligands, Gwt1 inhibitors, and prenylation inhibitors.
In Chapter 2, prenylation inhibition is investigated as a drug development strategy. Novel synthesized compounds and the Food & Drug Administration (FDA) approved carcinoma product Tipifarnib are assessed for their in vitro antifungal activity through microdilution broth minimum inhibitory concentration (MIC) assays. The mechanism of action of Tipifarnib was visualized using fluorescence microscopy before resistance mechanisms were interrogated by isolating fungal colonies with reduced tipifarnib susceptibility and genetically assessed mutations responsible for resistance.
In Chapter 3, the anticancer and immunosuppressive drug rapamycin is investigated for application as an antifungal drug. Treatment with rapamycin mimics starvation and induces cell death through autophagy. Using antifungal susceptibility MIC assays, rapamycin antifungal efficacy is shown to be significantly impacted by the nutritional environment. Additionally, synthesized analogs of rapamycin are investigated for their potential to specifically inhibit the fungal target of FK506 binding protein (FKBP12). Analog specificity is measured by comparing in vitro antifungal activity to immunosuppressive activity. A lead fungal specific analog is tested during an in vivo infection model demonstrating no adverse effects or therapeutic benefit at the concentrations tested.
In Chapter 4, immunosuppressive therapeutic agent FK520 is structurally modified and assayed for fungal specificity. Structure-guided drug design is used to develop two libraries of FK520 analogs. Through in vitro assessment of their antifungal and immunosuppressive activities, the lead compound JH-FK-08 is identified and used as an antifungal therapy during an in vivo cryptococcosis model. JH-FK-08 is shown to significantly reduce fungal burden and extend survival in addition to demonstrating a similar pharmacokinetic profile to parent compound FK506.
In Chapter 5, combination therapies are investigated for FK506 and JH-FK-08. Checkerboard assays are applied to test the interaction of two drugs when inhibiting fungal growth in vitro. JH-FK-08 is shown to have synergistic activity with Gwt1 inhibitors against C. neoformans and Candida albicans. FK506 is shown to have synergistic interactions with rapamycin which is unexpected due to the shared requirement of binding chaperone protein FKBP12. An FKBP12 mutant is shown to have no defect during virulence and competition in vivo experiments. This indicates that an FKBP12 mutation could confer resistance to a combination of FK506 and rapamycin.
Chapter 6 summarizes the results presented throughout this dissertation. The conclusions, impact of findings, and future directions are further considered within the broader context of antifungal therapeutic approaches. In addition, the benefits of repurposing established medications for novel use are highlighted and discussed here. Targeting proteins found in both humans and fungi can be an effective approach especially when designing structurally specific fungal inhibitors. This thesis demonstrates the efficacy of this approach through the development of promising antifungal agent JH-FK-08 shown to significantly extend survival and reduce fungal burden in mice undergoing an invasive fungal infection.
Item Open Access Genome evolution in the fungal pathogen Cryptococcus deuterogattii(2017) Billmyre, Robert BlakeOne of the key challenges of the 21st century is the emergence and reemergence of pathogens. Fungal pathogens represent an important portion of this problem, as the cohort of immunocompromised patients susceptible to common fungal pathogens rapidly expands in the developed world, and HIV/AIDS continues to present substantial challenges in the developing world. Understanding the processes by which pathogens emerge is critical, both for treating current outbreaks and for preventing future outbreaks. Here I have focused on the human fungal pathogen Cryptococcus deuterogattii, an emerging pathogen responsible for an ongoing outbreak in the Pacific Northwest region of the United States and Canada over the past approximately 15 years. To do so, I have taken comparative genomic, population genomic, and classic genetic approaches to understanding the origin of the outbreak, and the evolution of virulence at three different levels. I will first compare C. deuterogattii to the rest of the Cryptococcus pathogenic species complex, followed by comparisons of clonal clusters within C. deuterogattii, and finally I will compare individual strains within the VGIIa cluster.
I will begin in Chapter 1 by introducing genetic and non-genetic drivers of phenotypic diversity. I will then introduce the diversity of the fungal RNAi pathway, as well as discuss the frequent losses of the RNAi pathway throughout the eukaryotic kingdom.
In Chapter 2, I will examine the differences between the C. deuterogattii species responsible for the Pacific Northwest outbreak and the non-outbreak members of the Cryptococcus pathogenic species complex. I began by comparing the R265 reference genome of C. deuterogattii to the C. neoformans, C. deneoformans, and C. gattii reference genomes. Here we discovered that the core components of the RNAi pathway, including both argonautes, one of the two Dicers, and the only RNA-dependent RNA polymerase had been lost in the C. deuterogattii genome. These gene losses are conserved across the entire species and are defining characteristics of the species. We utilized this information to conduct a comparative genomics screen and identified a total of 14 conserved genes that were lost in the C. deuterogattii genome. We tested these for function in the RNAi pathway and discovered that a number of previously uncharacterized genes are novel RNAi components. In total, 9 of the 14 genes have been shown to play a role in the RNAi pathway. We demonstrated that the sex-induced (SIS) and mitotic-induced (MIS) silencing pathways share core components, and that SIS requires additional pathway components not required by MIS, suggesting that SIS may be a related and more specialized version of MIS. Finally, we showed that SIS appears to be induced through the pheromone signaling and MAP kinase cascade.
In Chapter 3, I will describe a whole genome resequencing project where we sequenced and analyzed clonal outbreak strains from the Pacific Northwest, related strains from outside the outbreak, as well as representatives of diverse global isolates. We utilized phylogenomic inference to provide evidence that the three clonal subgroups of the outbreak had distinct proximal origins: VGIIa in South America, VGIIb in Australia, and VGIIc with no identified origin outside the United States. We also demonstrated that the C. deuterogattii population shows patterns consistent with ancestral mating, but shows little evidence of more recent mating events, meaning that the population is characterized primarily by long periods of clonal growth with only intermittent episodes of sexual recombination.
In Chapter 4, I will examine variation within the VGIIa and neighboring VGIIa-like groups of C. deuterogattii uncovered through our resequencing study. We identified an msh2 nonsense allele ancestral to the VGIIa-like group. Here we demonstrate that this allele is responsible for a mutator phenotype that is particularly severe in genes containing homopolymer runs. Mutator strains are uncommon in eukaryotic microbes, and this lineage may represent a rare stable and successful environmental hypermutator lineage. However, I will also present evidence that the mutator state leads to high mutational burden and eventually loss of virulence, and argue that the mutator allele did not play a role in the expansion of the Pacific Northwest Outbreak in the VGIIa group.
In Chapter 5, I will conclude this thesis and provide some thought towards future directions that emerge from this work. Finally, in Appendices A and B I will discuss a pair of unfinished projects focusing on identifying novel mycoviruses in RNAi deficient lineages and the genetic basis of 5-FC resistance.
Item Open Access Global guideline for the diagnosis and management of rare mould infections: an initiative of the European Confederation of Medical Mycology in cooperation with the International Society for Human and Animal Mycology and the American Society for Microbiology.(The Lancet. Infectious diseases, 2021-08) Hoenigl, Martin; Salmanton-García, Jon; Walsh, Thomas J; Nucci, Marcio; Neoh, Chin Fen; Jenks, Jeffrey D; Lackner, Michaela; Sprute, Rosanne; Al-Hatmi, Abdullah MS; Bassetti, Matteo; Carlesse, Fabianne; Freiberger, Tomas; Koehler, Philipp; Lehrnbecher, Thomas; Kumar, Anil; Prattes, Juergen; Richardson, Malcolm; Revankar, Sanjay; Slavin, Monica A; Stemler, Jannik; Spiess, Birgit; Taj-Aldeen, Saad J; Warris, Adilia; Woo, Patrick CY; Young, Jo-Anne H; Albus, Kerstin; Arenz, Dorothee; Arsic-Arsenijevic, Valentina; Bouchara, Jean-Philippe; Chinniah, Terrence Rohan; Chowdhary, Anuradha; de Hoog, G Sybren; Dimopoulos, George; Duarte, Rafael F; Hamal, Petr; Meis, Jacques F; Mfinanga, Sayoki; Queiroz-Telles, Flavio; Patterson, Thomas F; Rahav, Galia; Rogers, Thomas R; Rotstein, Coleman; Wahyuningsih, Retno; Seidel, Danila; Cornely, Oliver AWith increasing numbers of patients needing intensive care or who are immunosuppressed, infections caused by moulds other than Aspergillus spp or Mucorales are increasing. Although antifungal prophylaxis has shown effectiveness in preventing many invasive fungal infections, selective pressure has caused an increase of breakthrough infections caused by Fusarium, Lomentospora, and Scedosporium species, as well as by dematiaceous moulds, Rasamsonia, Schizophyllum, Scopulariopsis, Paecilomyces, Penicillium, Talaromyces and Purpureocillium species. Guidance on the complex multidisciplinary management of infections caused by these pathogens has the potential to improve prognosis. Management routes depend on the availability of diagnostic and therapeutic options. The present recommendations are part of the One World-One Guideline initiative to incorporate regional differences in the epidemiology and management of rare mould infections. Experts from 24 countries contributed their knowledge and analysed published evidence on the diagnosis and treatment of rare mould infections. This consensus document intends to provide practical guidance in clinical decision making by engaging physicians and scientists involved in various aspects of clinical management. Moreover, we identify areas of uncertainty and constraints in optimising this management.Item Open Access Myosin V Function and Regulation in the Morphogenesis of Aspergillus Fumigatus(2018) Renshaw, HilaryOver the last decade, a growing number of fungal infections of animals and plants has risen and persisted. The lack of diversity in antifungal drugs as well as rising antifungal resistance in many pathogens has exacerbated the problem. Thus there is a critical need for basic molecular understanding of fungal morphogenesis and pathogenesis to design new ways to combat these diseases.
One of the fungal infections in need of new treatments is Aspergillus fumigatus, the etiological agent of invasive aspergillosis. A. fumigatus is an obligate filamentous fungus that is commonly found in the soil and air. It generally does not cause invasive disease in immunocompetent hosts; however immunocompromised people are at risk for invasive aspergillosis. To better understand the morphogenesis and pathogenesis of this fungus, I decided to study myosins, a group of actin-based motor proteins that are involved in myriad of integral processes in other organisms.
Through gene deletion, I revealed the importance of the class II myosin, MyoB, in septal formation and conidiation. The class V myosin, MyoE, was required for hyphal polarity, radial extension, septal frequency and conidiation. Importantly, MyoE was required for full virulence in a murine model of invasive aspergillosis. Given the importance of MyoE in critical processes such as hyphal growth and pathogenesis, I aimed to understand the molecular requirements of MyoE. Through iterative truncations of MyoE’s N-terminal tail domain, I revealed the importance of the tail domain in hyphal growth, polarity, and MyoE localization. I identified several phosphorylated residues on the MyoE protein, but mutational analysis did not reveal that any one residue was required for MyoE function. In the absence of the serine/threonine phosphatase, calcineurin, MyoE was phosphorylated at an additional residue. Mutational analysis of a residue in the tail domain revealed it was required for septal localization but not hyphal tip localization, growth, or septation.
Because MyoE is a cargo binding protein, it likely participates in several pathways that are required for growth and septation of the fungus. To identify novel roles of class V myosins, I identified the MyoE interactome using LC-MS/MS analysis. This analysis revealed several components of the COPII pathway for ER to Golgi transport, suggesting that MyoE may play a role in this protein transport system. My future work aims to understand this role.
Item Open Access Next-Generation Approaches to Understanding the Diversity and Evolution of Marine Fungi(2017) Picard, KathrynFungi are among the most diverse extant eukaryotic lineages, with estimates of total global diversity projecting millions of species that have yet to be cataloged. Though fungi from all phyla and habitats await discovery, marine fungi are particularly poorly understood. Historical surveys of fungi in marine habitats, which relied primarily on direct culturing or observation of fruiting bodies and other structures on incubated shore detritus, suggest that marine fungi are unexceptional in their diversity and frequency—and, therefore, unimportant, one might be tempted to conclude. However, with the increasing adoption of environmental sequencing as a primary tool for exploring fungal diversity and ecology across disparate habitats, the discovery of novel phylotypes representing new species—and in some cases, even new phyla—demands a reappraisal of fungal diversity in marine habitats using modern molecular methods. This dissertation represents an attempt to advance our understanding of the breadth of fungal diversity, to establish a broader evolutionary context for marine fungi, and to provide some molecular tools to better study marine fungi that have, until now, eluded our detection.
In Chapter 1, I investigate the diversity and spatio-temporal distribution of unicellular eukaryotes in the surface waters of the English Channel using high-throughput sequencing of 18S ribosomal DNA. In addition to characterizing the taxonomic and phylogenetic diversity of planktonic protists, I also estimate the niche breadth of the taxa observed and infer ecological roles and trophic modes to examine if and how functional guilds change through space and time. I find that while the community observed at any given time is likely to be dominated by only a handful of species, many of them members of the Stramenopiles, Alveolates, and Rhizaria, most protistan taxa are rare specialists. I also find that the relative abundance of an individual taxon is not indicative of a specialist or generalist habit. Interestingly, I also find that fungi comprise a significant fraction of the microbial community, but the most prominent fungal taxa observed do not closely resemble phylogenetically any circumscribed species of marine fungi. Rather, they are nested within the enigmatic Cryptomycota, a recently described phylum at the base of the fungal tree. Thus, while this is the only chapter of my dissertation which does not focus primarily on fungal diversity or evolution, the results in many ways set the stage for my subsequent work.
In Chapter 2, I turn my sails back toward land to challenge long-standing mycological lore by re-examining the diversity of coastal marine fungi. I focus on four marine habitats in coastal North Carolina (surface water, persistent wetlands, intertidal sand flats, and marine benthos) from which I sample water and sediments over the course of a year. Using primers designed to amplify across the entire fungal kingdom, I use the Ion Torrent platform to sequence amplicons from 28S ribosomal DNA and evaluate how successfully extant reference databases identify novel fungal sequences to both high and low taxonomic ranks. I find that marine fungi are far more varied than previously thought, with some early diverging fungi, and the Chytridiomycota in particular, proving to be diverse and ubiquitous. I also find that curated reference databases struggle to assign robust taxonomic identities to novel sequences across all fungal phyla, but are particularly ill-equipped to identify the marine representatives of non-Dikarya fungi.
In Chapter 3, I aim to address the deficiencies of curated reference databases used in taxonomic assignment by generating high-quality reference sequences from complex environmental samples. Using a third-generation sequencing technology, PacBio circular consensus sequencing, which trades the high coverage of other sequencing platforms for much longer read lengths, I target an approximately 2kb fragment of the ribosomal DNA operon that contains both the full fungal internal transcribed spacer (ITS) region and over 1kb of the 28S ribosomal subunit. Using a mock community approach and successive rounds of filtering, I calculate the average sequencing error for PacBio amplicons by comparing them to known sequences from axenically cultured, circumscribed species. I then revisit my samples from Chapter 2, generate amplicon sequences to be used for phylogenetic inference, and compare the accuracy of taxonomic assignments made by reference databases curated for individual loci (i.e., ITS vs. 28S) for different rDNA regions from the same operational taxonomic unit. I find that stringent quality filtering of PacBio sequence data produces consensus sequences approaching the quality of MiSeq and 454, which can be used in phylogenetic analyses to provide improved taxonomic assignments. Furthermore, many of the fungal taxa observed belong to known marine lineages, while others are only distantly related to reference accessions and may represent new lineages.
Item Open Access Novel Targets and Therapeutic Strategies for the Treatment of Cryptococcus neoformans(2024) Palmucci, Julia RoseInvasive fungal infections cause significant worldwide morbidity, and mortality rates remain remarkably high despite treatment. One of the most pressing fungal pathogen threats is the opportunistic infection, Cryptococcus neoformans. C. neoformans is a yeast found ubiquitously throughout the environment that can cause cryptococcal meningitis, primarily in immune suppressed individuals. Treatments for C. neoformans and other invasive fungal pathogens have a number of disadvantages. Currently, there are only four classes of antifungals available for treatment, and each has varying efficacy and toxicity profiles. Given the overlap in eukaryotic cellular machinery between fungal pathogens and their human host, many of the most effective treatments have significant associated toxicities, side-effects, and drug interactions. Additionally, many antifungal drugs are unavailable or inaccessible to the patient populations in greatest need. Therefore, novel therapeutic strategies and antifungal targets are urgently needed to address the threat of invasive fungal infections. My thesis work has subsequently focused on investigating novel treatment strategies, potential druggable targets, and novel antifungal compounds to combat C. neoformans and other invasive fungal pathogens.Given the limited options available for the treatment of invasive fungal infections, we employed a creative strategy to evaluate the effect of diet on antifungal drug efficacy. We determined that a high-fat, low-carbohydrate, adequate protein ketogenic diet potentiated the antifungal effect of fluconazole in vivo against murine models of systemic C. neoformans and Candida albicans infection. Pharmacokinetic/pharmacodynamic analysis revealed that a ketogenic diet significantly enhanced drug exposure in both blood plasma and brain tissue. However, this pharmacokinetic enhancement was not exclusively responsible for observed effect on tissue fungal burden, indicating a yet-unknown mechanism of fluconazole activity enhancement. We further evaluated the effects of a ketogenic diet on the immune system, the effect of ketone bodies on C. neoformans, and the activity of other antifungal compounds and diets. While the mechanism remains elusive, our findings indicate that a ketogenic diet potentiates the activity of fluconazole against C. neoformans and C. albicans at multiple body sites of infection. These results may have promising practical treatment implications in the future. C. neoformans is characterized by a strong brain tropism that has largely confounded researchers, as the brain traditionally presents a hostile, nutrient poor, and oxidatively stressful environment. However, C. neoformans is adept at establishing infection at this site. We asked whether nitrogen metabolism may play a key role in C. neoformans infection, especially within the central nervous system. Through RNA-seq analysis from Cryptococcus isolates derived directly from patient cerebrospinal fluid, we identified that genes encoding enzymes surrounding glutamine and glutamate metabolism were highly upregulated during infection. In particular, glutamine synthetase was found to be one of the most highly expressed genes of the entire Cryptococcus genome during central nervous system infection. We subsequently investigated the plasticity of this central nitrogen hub that interconverts glutamate and glutamine through the construction of multiple gene deletion mutations of its core enzymes. Surprisingly, while C. neoformans could tolerate loss of multiple enzymes in this central nitrogen hub, we determined that the glutamine synthetase enzyme is likely essential. Further work utilizing an inducible promoter system and known glutamine synthetase inhibitors supported this hypothesis. We finally evaluated a toxic analog of glutamine—the enzymatic product of glutamine synthetase, in an in vivo model of systemic C. neoformans infection. Taken together, we determined that glutamine synthetase is essential in C. neoformans, and glutamine metabolism may serve as a promising and novel druggable target for future antifungal drug development. I have next presented an overview of the opportunities for repurposing anticancer drugs to treat fungal infections and introduce two novel antifungal compounds currently in preclinical development. I have additionally provided a summary of findings presented in this dissertation, as well as several avenues for future research. Collectively, my thesis work has highlighted several novel strategies, targets, and compounds for future antifungal therapeutic development, and these studies emphasize the need for creativity and interdisciplinary thinking in the development of antifungal therapies.
Item Open Access Risk factors and outcome of pulmonary aspergillosis in critically ill coronavirus disease 2019 patients-a multinational observational study by the European Confederation of Medical Mycology.(Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases, 2022-04) Prattes, Juergen; Wauters, Joost; Giacobbe, Daniele Roberto; Salmanton-García, Jon; Maertens, Johan; Bourgeois, Marc; Reynders, Marijke; Rutsaert, Lynn; Van Regenmortel, Niels; Lormans, Piet; Feys, Simon; Reisinger, Alexander Christian; Cornely, Oliver A; Lahmer, Tobias; Valerio, Maricela; Delhaes, Laurence; Jabeen, Kauser; Steinmann, Joerg; Chamula, Mathilde; Bassetti, Matteo; Hatzl, Stefan; Rautemaa-Richardson, Riina; Koehler, Philipp; Lagrou, Katrien; Hoenigl, Martin; ECMM-CAPA Study GroupObjectives
Coronavirus disease 2019 (COVID-19) -associated pulmonary aspergillosis (CAPA) has emerged as a complication in critically ill COVID-19 patients. The objectives of this multinational study were to determine the prevalence of CAPA in patients with COVID-19 in intensive care units (ICU) and to investigate risk factors for CAPA as well as outcome.Methods
The European Confederation of Medical Mycology (ECMM) conducted a multinational study including 20 centres from nine countries to assess epidemiology, risk factors and outcome of CAPA. CAPA was defined according to the 2020 ECMM/ISHAM consensus definitions.Results
A total of 592 patients were included in this study, including 11 (1.9%) patients with histologically proven CAPA, 80 (13.5%) with probable CAPA, 18 (3%) with possible CAPA and 483 (81.6%) without CAPA. CAPA was diagnosed a median of 8 days (range 0-31 days) after ICU admission predominantly in older patients (adjusted hazard ratio (aHR) 1.04 per year; 95% CI 1.02-1.06) with any form of invasive respiratory support (HR 3.4; 95% CI 1.84-6.25) and receiving tocilizumab (HR 2.45; 95% CI 1.41-4.25). Median prevalence of CAPA per centre was 10.7% (range 1.7%-26.8%). CAPA was associated with significantly lower 90-day ICU survival rate (29% in patients with CAPA versus 57% in patients without CAPA; Mantel-Byar p < 0.001) and remained an independent negative prognostic variable after adjusting for other predictors of survival (HR 2.14; 95% CI 1.59-2.87, p ≤ 0.001).Conclusion
Prevalence of CAPA varied between centres. CAPA was significantly more prevalent among older patients, patients receiving invasive ventilation and patients receiving tocilizumab, and was an independent strong predictor of ICU mortality.