Browsing by Author "Alspaugh, J Andrew"

My thesis work has focused on characterizing mechanisms by which human fungal pathogens regulate their adaptive cellular responses in order to survive and cause disease in the human host. Unlike most microbial fungi found in the environment, Cryptococcus neoformans has become a successful human pathogen due to two intrinsic abilities: 1) to survive and grow at human body temperature and 2) to employ virulence factors to combat host immune defenses. Over the past two decades, the fungal pathogenesis field has made enormous progress in identifying and characterizing C. neoformans proteins responsible for these adaptive cellular responses with a particular focus on enzymes, like those involved in cell cycle progression or those responsible for synthesizing components of the fungal cell surface. Although we know a substantial amount about the functions of these enzymes and their implications on fungal pathogenesis, the mechanisms by which these enzymes are regulated are less clear. I have attempted to address this gap in knowledge by focusing my thesis work on the identification and characterization of C. neoformans non-enzymatic proteins that regulate enzymes important for adaptive cellular responses. I have identified and characterized the C. neoformans arrestin proteins as regulators of enzyme ubiquitination, and likely enzyme function, in response to specific extracellular stressors (Chapters 2 & 3). I have also characterized a Cryptococcus-specific protein, Mar1, as an important modulator of host-fungal interactions due to its regulation of cell surface remodeling through maintenance of mitochondrial metabolic activity and homeostasis in response to cellular stress (Chapters 4 & 5). Furthermore, I also performed a comprehensive comparative analysis of different RNA enrichment methods for RNA sequencing applications and long non-coding RNA identification in C. neoformans, which can help researchers select appropriate tools for studying adaptive cellular responses from the RNA level (Chapter 6). These studies collectively have demonstrated that non-enzymatic proteins are important “cellular coordinators” in human fungal pathogens; they regulate the activity of many different enzymes in response to distinct extracellular signals, and as a result are required for both fungal growth and virulence factor employment in response to host-relevant stressors.

Cryptococcus neoformans is a human fungal pathogen that is also ubiquitous in the environment. To cause disease inside a human host, C. neoformans must be able to sense and respond to a multitude of stresses. One of the major responses to the host is the induction of a polysaccharide capsule, which allows the fungus to resist damage and evade the host immune response. This capsule is regulated by a number of signal transduction cascades, but a major contributor is the conserved cAMP/PKA pathway.

Using genetic and molecular biology techniques, I identified Gcn5 and Rim101 as key transcriptional regulators of capsule within the host. I determined that C. neoformans Rim101 is activated by a combination of the canonical pH sensing pathway and the cAMP/PKA pathway. This novel connection potentially gives the pathogen greater flexibility in responding to environmental stimuli, thus allowing for a greater capacity for disease.

I determined that the Rim101 transcription factor regulates cell wall remodeling in the context of the host by deep mRNA sequencing, electron microscopy, and biochemical assays. Using chromatin immunoprecipitation, I confirmed that these cell wall changes are under direct control of Rim101. I then confirmed the importance of cell wall changes in the host by nanoString profiling of fungal RNA in the context of a murine lung infection. I also examined the lungs of infected mice for cytokine and immune cell infiltrate and determined that C. neoformans cell wall changes are important in avoiding triggering an aberrant host response. I hypothesize that this cell wall remodeling via Rim101 activation is required for full capsule attachment and for masking immunogenic molecules from the host immune system.

Stress tolerance and adaptability to dynamic environments are two things that make a microbial pathogen especially dangerous in the setting of a human infection. Cryptococcus neoformans, a ubiquitous pathogenic fungus, is able to sense, adapt, and tolerate the stressful environment of the human host in order to survive and cause disease. From the time this pathogen is inhaled into the lung to when it enters the central nervous system to cause life-threatening cryptococcal meningoencephalitis, C. neoformans activates numerous stress response signaling pathways to convert extracellular cues into adaptive cellular responses to ensure its survival in a new environment. Upon entering the human host, C. neoformans must overcome the stress of increased extracellular pH in order to survive. This organism is naturally found in environmental reservoirs with a pH of 5-6, but must adapt to a relatively alkaline pH pf 7.4 in niches of the human host such as the blood stream and interstitial alveolar space. Our work focuses on the ability for this fungal pathogen to modify both its cell wall and cell membrane using pH-response signaling pathways in order to thrive in an alkaline environment. Elucidating the mechanism of this pH response will not only help us understand the way this particular pathogen adapts to novel environments, but also reveal how we might manipulate certain components or processes in these adaptive signaling pathways to prevent and treat this invasive fungal infection. One example of a known external pH-sensing process in many model fungi and fungal pathogens is the Rim/Pal signal transduction pathway. Mutations in this pathway result in strains that are attenuated for survival at alkaline pH, and often for survival within the host due to the role for this pathway in cell wall remodeling and maintenance. We used an insertional mutagenesis screen to identify novel upstream components in the Rim pathway required for C. neoformans growth at host pH. We discovered altered alkaline pH growth in several strains with specific defects in plasma membrane composition and maintenance of phospholipid assembly. Among these, loss of function of the Cdc50 lipid flippase regulatory subunit affected the temporal dynamics of Rim pathway activation. Lipid flippase complexes, including Cdc50, are essential for maintaining the asymmetric distribution of phospholipids in the plasma membrane. We explored how Cdc50-mediated maintenance of lipid asymmetry affect membrane-bound pH-sensing proteins in the Rim pathway to facilitate signaling. Specifically, we demonstrated how the upstream Rim pathway activator and pH sensor, Rra1, uses its C-terminal tail to sense these alterations in lipid asymmetry and activate the downstream portion of the pathway. These results suggest both broadly applicable and phylum-specific molecular interactions that drive microbial environmental sensing involving the Rim alkaline response pathway. The ability for cells to internalize extracellular cues allows them to adapt to novel and stressful environments. The Rim pathway effectively converts the extracellular signal of increased pH into an adaptive cellular response allowing the pathogen to survive in its new environment. As previously mentioned, Rra1 is a plasma membrane protein responsible for sensing and internalizing the alkaline pH signal. We further identify the specific mechanisms of Rim pathway signaling through detailed studies of the activation of the Rra1 protein. Specifically, we observe that the Rra1 protein is internalized and recycled in a pH-dependent manner and that this further depends on specific residues on its C-terminal tail, clathrin-mediated endocytosis, and the integrity of the plasma membrane. These results continue to unravel the complex and intricate dynamics of membrane-mediated pH-sensing in a relevant human fungal pathogen. Observations from our genetic screen revealed that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. We find that an elevated pH is sufficient to induce activation of the sterol homeostasis pathway transcription factor, Sre1. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol, but is not dependent on Rim pathway signaling, indicating that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals under alkaline conditions, highlighting the impact of microenvironmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response. Rim-mediated modifications of both fungal cell wall components and membrane lipids combined with the ergosterol essentiality in the ability for fungal cells to grow in alkaline environments led us to explore the cell exterior in more detail. We include a comprehensive review of what is currently known in the field about the backbone structures of the cell wall: chitin and chitosan. A greater understanding of the complex layering that composes the structures connected to the plasma membrane will elucidate the barrier function these components provide in the collective response to pH stress. These studies revealing exploring the mechanisms of the alkaline pH response in a relevant human fungal pathogen will enhance our understanding of how these microorganisms tolerate and overcome the stressful host environment. Furthermore, the fact that these alkaline signaling pathways intimately involve the dynamics of the plasma membrane, further elucidate the general mechanisms by which cells respond to and internalize changes in extracellular environments using the exterior architecture of the cell.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease and death in immunocompromised populations, in particular among those with advanced HIV infection. This fungus is found ubiquitously in the environment and acquired through inhalation into the respiratory tract followed by dissemination to the central nervous system in immunocompromised individuals. The ability for C. neoformans to sense and adapt to the host environment is crucial to its success as a pathogen. Many C. neoformans proteins require proper subcellular localization for their function, and as such this fungus carefully regulates the localization of proteins involved in important cellular processes related to host adaptation.

Fungal growth and morphogenesis, as well as thermotolerance and virulence are controlled by conserved Ras-like GTPases. These proteins require proper localization for full function and are directed to cellular membranes through the posttranslational modification process known as prenylation. Using the tools of fungal genetics and molecular biology, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase -subunit is required for thermotolerance and pathogenesis. We also identified and characterized post-prenylation protease and carboxyl methyltransferase enzymes in C. neoformans, demonstrating that these later steps have only subtle effects on stress response and fungal virulence. By fluorescent microscopy and molecular biology, we show that Ram1 is required for proper subcellular localization of Ras1, but not Cdc42, and that the post-prenylation processing steps are dispensable for the localization of these substrate proteins.

C. neoformans dramatically alters its cell wall upon entering the host in order to facilitate immune avoidance. Using the tools of forward genetics, we identified a novel cell wall regulatory protein, Mar1. We have demonstrated that this protein is required for capsule attachment and full virulence in mouse models of infection. Using staining and biochemical techniques, we have characterized the cell wall of mar1∆ mutant cells, and by fluorescent microscopy we have demonstrated that the -(1,3)-glucan synthase catalytic subunit, Fks1, is mislocalized in mar1∆ cells. Using in vitro co-culture models, we have determined that the mar1∆ cell wall induces increased macrophage activation that is dependent on the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR-2 pattern recognition receptors.

To further understand the impact of the Mar1 protein on the host-pathogen interaction, we used in vivo mouse models to characterize the pathogenesis and immune response to this strain. Using histopathology and light microscopy, we have shown that mar1∆ cells induce granulomas in the lungs of infected mice, an in in vitro co-culture models we have demonstrated that the mar1∆ strain induces increased markers angiogenesis. Finally using immunization strategies, we show that the mar1∆ strain does not induce a protective response against a secondary lethal challenge.

Lastly, using bioinformatics tools and batch sampling, we developed a novel computational tool to more efficiently analyze mutants of interest generated by forward genetic screens. We demonstrate the efficacy of this tool through proof of principle experiments that led us to the discovery of the Mar1 protein described above. Additional projects in our lab and others have already utilized this mutant analysis tool in C. neoformans and we propose that it can be ultimately applied to a wide range of experimental systems and methods of mutagenesis, facilitating future microbial genetic screens.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH.

Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms.

The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi.

To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.

Cryptococcus neoformans pathogenesis results from the proliferation of yeast-phase fungal cells within the human host. The Ras1 signal transduction cascade is a major regulator of C. neoformans yeast and hyphal-phase morphogenesis, thermotolerance, and pathogenesis. Previous work identified the conserved Rho-GTPases Cdc42 and Rac1 as potential downstream targets of Ras1. In this work, we identify the duplicate Cdc42 and Rac paralogs, Cdc42 and Cdc420, and Rac1 and Rac2, as major effectors of Ras1-mediated thermotolerance and polarized growth, respectively. Using genetic and molecular biology techniques, including mutant analyses and over-expression studies, we determine the separate and overlapping roles of the four Rho-GTPases in Ras1-mediated morphogenesis. The Cdc42 paralogs are non-essential but are required for thermotolerance and pathogenesis. Ras1 acts through the Cdc42 paralogs to regulate cytokinesis via the organization of septin proteins. The major paralog, Cdc42, and the minor paralog, Cdc420, exhibit functional differences that are primarily dictated by transcriptional regulation. Additionally, CDC42 transcription is induced by exposure to temperature stress conditions. In contrast, Ras1 acts through the equivalently transcribed RAC paralogs to regulate polarized growth during both yeast and hyphal-phase morphogenesis. Rac1 and Rac2 are individually dispensable and appear to be functionally redundant but are synthetically required for yeast phase growth and spore development. The sub-cellular localization of the Rac paralogs is dependent on both Ras1 and post-translational modification by prenyl transferases. The identification and characterization of the conserved elements of the Ras1 signal transduction cascade presented here constitute an important contribution towards the design of anti-fungal agents that are based on existing Ras-pathway inhibitors.

The fungal pathogens encounter ever-changing conditions during their pathogenic life cycles, including shifts from the environment to the human host. Fungi have evolved many pathways that allow them to overcome and even thrive in the presence of the stresses presented by different environments.

Antifungal drug treatment presents a significant stress for the opportunistic pathogen Cryptococcus neoformans. Currently, there are limited treatment options for cryptococcal infections. Researchers have been working toward identifying drugs that inhibit fungal-specific processes, such as the echinocandins, which target the synthesis of the fungal cell wall component β-1,3-glucan. However, C. neoformans is highly tolerant of these drugs, despite their effective inhibition of the sole and essential cryptococcal β-1,3-glucan synthase. We therefore performed a screen through a deletion mutant collection to identify compensatory processes involved with echinocandin tolerance. In this way, we identified several processes that are required for full tolerance of these drugs, including stress-induced responses and cell wall biosynthesis. Overall, these studies implicate distinct and targetable cellular processes that might be exploited to enhance echinocandin efficacy for treating infections caused by C. neoformans.

An alteration in extracellular pH is another one of the predictable changes that C. neoformans encounters in its shift from its environmental niche to the human host. This environmental increase in pH is internalized into microbial cells through the fungal-specific Rim signal transduction pathway. We have determined that the upstream pH-sensing components of Rim signaling are significantly divergent in the basidiomycete phylum, which includes C. neoformans, agricultural pathogens, and saprophytes. Recently, we identified the first basidiomycete Rim pathway pH sensor, the C. neoformans Rra1 protein.

Through a proteomics-based screen for potential Rra1 pH sensor signaling partners or interactors, we identified nucleosome assembly protein 1 (Nap1) as an interactor of the Rra1 pH sensor. Like Rra1, C. neoformans Nap1 is required for the activation of the Rim pathway. Nap1 specifically interacts with the Rra1 protein, acting as a scaffold to maintain stability of the Rra1 pH sensor protein in the cell. In current work, we are exploring how Nap1 and other proteins regulate the fungal pH sensing complex, through both localization and post-translational modification of the Rra1 pH sensing protein.

Finally, I expanded these studies into another basidiomycete fungus, the skin commensal and pathogen, Malassezia sympodialis. In this fungus, I confirmed that the Rra1 pH sensor is, in fact, a basidiomycete-specific protein and that a functional Rim pathway is required for growth at high pH or salt concentrations. Finally, through RNA-sequencing analyses, we identified genes that are regulated by the Rim pathway in response to alkaline pH. These studies have expanded our knowledge about Rim pathway function in basidiomycete fungi.

Together these inter-related experimental approaches explore ways in which C. neoformans adapts to overcome and survive stressful environments. We have identified novel signaling elements of conserved, stress-response pathways in fungi. Additionally, we have explored mechanisms by which important human pathogens display intrinsic tolerance to established antifungal agents, providing insight into potential new avenues for antimicrobial therapy.