Browsing by Subject "Fungal pathogenesis"

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.

Fungal cells sense the amount and quality of external nutrients through multiple interconnected signaling networks, which allow them to adjust their metabolism, transcriptional profiles and developmental programs to adapt readily and appropriately to changing nutritional states. In organisms ranging from yeasts to humans, the Tor signaling pathway responds to nutrient-derived signals and orchestrates cell growth. While in the baker's yeast Saccharomyces cerevisiae Tor responds to nutrient-derived signals and orchestrates cell growth and proliferation, in Schizosaccharomyces pombe Tor signaling modulates sexual differentiation in response to nutritional cues. Thus, these differences provide a framework to consider the roles of Tor in other fungal organisms, in particular those that are pathogens of humans.

In this dissertation, I demonstrate that in the human fungal pathogen Candida albicans, Tor signaling also functions to promote growth. This study also uncovered a novel role for the Tor molecular pathway in promoting hyphal growth of C. albicans on semi-solid surfaces and in controlling cell-cell adherence. Gene expression analysis and genetic manipulations identified several transcriptional regulators (Bcr1, Efg1, Nrg1, and Tup1) that together with Tor compose a regulatory network governing adhesin gene expression and cellular adhesion. While the Tor kinases are broadly conserved, these studies further demonstrate the contrasting strategies employed by fungal organism in utilizing the Tor signaling cascade.

While extensive studies have focused on elucidating functions for the Tor signaling cascades among ascomycetes, little is known about the pathway in basal fungal lineages, in particular among zygomycetes and chytrids. Moreover, given that the Tor pathway is the target of several small molecule inhibitors including rapamycin, a versatile pharmacological drug used in medicine, there is considerable interest in Tor signaling pathways and their function. Capitalizing on emerging genome sequences now available for several basal fungal species, we show a remarkable pattern of conservation, duplication, and loss of the Tor signaling cascade among basal fungal lineages. Targeting the pathway with rapamycin results in growth arrest of several zygomycete species, indicating a conserved role for this pathway in regulating fungal growth. In addition, we show a potential therapeutic advantage of using rapamycin in a heterologous model of zygomycosis. Taken together, the Tor signaling cascade and its inhibitors provide robust platforms from which to develop novel antimicrobial therapies, which may include less immunosuppressive rapamycin analogs.