Interaction of Cryptococcus neoformans Rim101 and protein kinase A regulates capsule.

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101 multiply sign in circle mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101 multiply sign in circle strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101 multiply sign in circle mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions.

Drivers of Dengue Within-Host Dynamics and Virulence Evolution

Dengue is an important vector-borne virus that infects on the order of 400 million individuals per year. Infection with one of the virus's four serotypes (denoted DENV-1 to 4) may be silent, result in symptomatic dengue 'breakbone' fever, or develop into the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Extensive research has therefore focused on identifying factors that influence dengue infection outcomes. It has been well-documented through epidemiological studies that DHF is most likely to result from a secondary heterologous infection, and that individuals experiencing a DENV-2 or DENV-3 infection typically are more likely to present with more severe dengue disease than those individuals experiencing a DENV-1 or DENV-4 infection. However, a mechanistic understanding of how these risk factors affect disease outcomes, and further, how the virus's ability to evolve these mechanisms will affect disease severity patterns over time, is lacking. In the second chapter of my dissertation, I formulate mechanistic mathematical models of primary and secondary dengue infections that describe how the dengue virus interacts with the immune response and the results of this interaction on the risk of developing severe dengue disease. I show that only the innate immune response is needed to reproduce characteristic features of a primary infection whereas the adaptive immune response is needed to reproduce characteristic features of a secondary dengue infection. I then add to these models a quantitative measure of disease severity that assumes immunopathology, and analyze the effectiveness of virological indicators of disease severity. In the third chapter of my dissertation, I then statistically fit these mathematical models to viral load data of dengue patients to understand the mechanisms that drive variation in viral load. I specifically consider the roles that immune status, clinical disease manifestation, and serotype may play in explaining viral load variation observed across the patients. With this analysis, I show that there is statistical support for the theory of antibody dependent enhancement in the development of severe disease in secondary dengue infections and that there is statistical support for serotype-specific differences in viral infectivity rates, with infectivity rates of DENV-2 and DENV-3 exceeding those of DENV-1. In the fourth chapter of my dissertation, I integrate these within-host models with a vector-borne epidemiological model to understand the potential for virulence evolution in dengue. Critically, I show that dengue is expected to evolve towards intermediate virulence, and that the optimal virulence of the virus depends strongly on the number of serotypes that co-circulate. Together, these dissertation chapters show that dengue viral load dynamics provide insight into the within-host mechanisms driving differences in dengue disease patterns and that these mechanisms have important implications for dengue virulence evolution.