Browsing by Subject "Aspergillus fumigatus"

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Although improvements in antifungal therapy have advanced the treatment of cryptococcosis, the mortality rate is approximately 12% in medically advanced countries, and approaches 50% in less developed regions. Additionally, C. neoformans can cause infection in seemingly healthy individuals, elevating its status as a primary human pathogen. Although numerous studies have examined virulence properties, less is understood regarding host immune factors in the lungs during early stages of fungal infection. In the present thesis studies, I examined the roles played by pulmonary surfactant proteins in response to C. neoformans in vitro and in vivo. We demonstrate that SP-D, but not SP-A, binds to the yeast and increases phagocytosis of poorly encapsulated yeast cells by macrophages, yet concomitantly protects the pathogenic microbes from macrophage-mediated defense mechanisms. Furthermore, we show that SP-D functions as risk factor in vivo by protecting the yeast cells against oxidant species and thus facilitating disease progression. The results of these studies provide a new paradigm on the role played by surfactant protein D during host responses to C. neoformans and, consequently, impart insight into potential future treatment strategies for cryptococcosis.

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Over 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.

Aspergillus fumigatus, the main etiology of invasive aspergillosis, is a leading cause of fungal mortality in immunocompromised patients. Although the incidence of invasive aspergillosis has increased in the last two decades due to a rise in the immunocompromised patient population, there is a lack of effective treatments and basic understanding of growth and disease. Septins are conserved GTPases that are involved in a myriad of cellular processes, ranging from cytokinesis to cell morphology. Here we describe the role of septins in A. fumigatus growth, development, and pathogenesis. Through gene deletion, we revealed that all 5 septin genes are dispensable for growth under basal conditions. Nonetheless, AspA, AspB, AspC, and AspE are required for regular septation and the core septins are required for conidia production. The ΔaspB strain was hypervirulent in the Galleria mellonella model of infection, while virulence was similar to the wild-type strain in our murine model of invasive aspergillosis. Deletion of aspB also lead to an increase in susceptibility to anti-cell wall agents. AspB, which under basal conditions interacts with 334 proteins, alters its protein interaction after exposure to a clinically relevant concentration of the antifungal caspofungin. A total of 69 of the basal interactants do not interact with AspB, and 54 new interactants were identified following caspofungin exposure. Of the interactants studied, only PpoA was implicated in the response to anti-cell wall agents.

Due to the pleiotropic role of AspB, we characterized how posttranslational modifications regulate AspB function. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension. Gin4, Cla4, and ParA contributes to both septation and conidiation of A. fumigatus. Deletion of cla4 and parA lead to hyperseptation, while deletion of gin4 leads to larger apical compartments that were similar in length to the ΔaspB strain. Cla4, Gin4, and ParA contribute to proper localization of AspB. Phosphoproteomic analyses show that AspB is phosphorylated at 7 residues: 5 in the GTPase domain, and 2 at C-terminus. Deletion of gin4 and cla4 did not alter the phosphorylated residues in AspB. Deletion of parA results in 2 new phosphorylation sites identified, one in the N-terminal polybasic region (T68) and one in the coil-coiled domain (S447). Mutation of T68 to glutamic acid leads to a slight increase in interseptal distances. These results demonstrate the key role of septins in response to anti-cell wall agents, as well as how phosphorylation regulates septin function in A. fumigatus.