Frawley, Dean (2020) The Roles of a Conserved MAP Kinase Pathway in the Regulation of Development and Secondary Metabolism in Aspergillus Species. PhD thesis, National University of Ireland Maynooth.

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Abstract

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signalling pathways are utilised. An example of highly conserved signalling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. MAPK pathways are responsible for the regulation of a diverse array of biological processes, such as cell proliferation, immune responses and metabolism, to name a few. In fungal organisms, MAPK pathways have been shown to be involved in the modulation of cell fusion and mating, asexual and sexual reproduction, cellular stress responses and secondary metabolism. MAPK pathways have been extensively studied in yeast, a model organism for unicellular fungi. One MAPK pathway in particular is known as the pheromone module, which consists of three kinases (Ste11, Ste7, Fus3), the adaptor protein Ste50 and the scaffold protein Ste5. This pathway is critical for the response to pheromone signalling between neighbouring yeast cells, resulting in the regulation of cell fusion, otherwise known as mating or sexual reproduction. Orthologous MAPK pathways have been studied in fungal species such as the model ascomycete Neurospora crassa. In this species, the pheromone module pathway has been shown to be involved in the regulation of germling and hyphal fusion, which is critical for the establishment of the interconnected fungal mycelium. With respect to the genus Aspergillus, information regarding the influence of MAPK signalling pathways in the regulation of fungal development is limited. Orthologs of the core pheromone module components in yeast have been identified previously in the model filamentous fungus A. nidulans. These include the three kinases SteC, MkkB and MpkB (orthologs of yeast Ste11, Ste7 and Fus3 respectively) and the adaptor protein SteD (ortholog of yeast Ste50). However, there are no orthologs of the Ste5 scaffold in filamentous fungal genomes, suggesting a unique mechanism of regulation exists for the pheromone module pathway in species like A. nidulans. This led to the first aim of this research thesis which was to identify and characterise a scaffold candidate for the pheromone module in A. nidulans. We identified the AN2701 gene which encodes a large protein consisting of multiple WD40 repeats, which is characteristic of scaffold proteins. This gene is orthologous to the N. crassa ham5 gene which encodes a scaffold protein in the Mak-2 pheromone module pathway. For this reason, we named the protein product of the AN2701 gene ‘HamE’. Via a genetic and proteomic approach, we provided evidence that the HamE scaffold physically interacts with the kinases MkkB and MpkB and that a pentameric complex is formed at the hyphal tips and plasma membrane. HamE is essential for the regulation of kinase phosphorylation levels and a hamE mutant exhibits dramatically reduced levels of MpkB phosphorylation, signifying reduced MAP kinase signalling and transcription factor activation. Deletion of any of the five members of the complex results in reductions in asexual sporulation, as well as complete inhibition of sexual cleistothecia formation and dramatic decreases in the expression levels of various secondary metabolite genes. The next aim of this research thesis was to further characterise the roles of the pheromone module in Aspergillus species. Due to the dramatic defects in development and secondary metabolism observed in pheromone module mutants in A. nidulans, it was decided to assess whether the pheromone module could be utilised by other Aspergillus species to regulate their development and secondary metabolism. We found that orthologs of the pheromone module genes are highly conserved in the genus Aspergillus. We identified orthologs of each of the five core pheromone module components in two pathogenic fungi, A. flavus and A. fumigatus. A. flavus is a prolific producer of secondary metabolites such as the highly carcinogenic compound aflatoxin B1. This species is considered a major global threat as it is capable of causing contamination of a wide variety of agricultural crops and ingestion of crops contaminated with aflatoxin can lead to the development of hepatocellular carcinomas. A. fumigatus is an opportunistic human pathogen and is a major risk to immunocompromised individuals. This species produces dangerous secondary metabolites such as the immunosuppressant gliotoxin, which promotes invasive pulmonary aspergillosis and this can lead to mortality rates as high as 95% in immunocompromised patients. In A. flavus, we provided evidence of the existence of a tetrameric pheromone module pathway, which is made up of two sub-complexes. Initially, MkkB and MpkB form a dimer at the hyphal tips, potentially in response to chemotropic interactions between neighbouring hyphae. The SteC-SteD dimer then interacts with the MkkBMpkB dimer in the cytoplasm and assembly of the complex results in phosphorylation and migration of MpkB into the nucleus, where it presumably interacts with various transcription factors. Interestingly, HamE was shown to localise to the hyphal tips but was not observed to interact with the pheromone module components, signifying a potentially unique mechanism of signalling in this species. Deletion of steC, mkkB, mpkB or steD results in inhibition of both asexual sporulation and sclerotia production. Additionally, each of these mutants produced significantly lower levels of aflatoxin B1 and increased levels of other secondary metabolites, such as leporin B and cyclopiazonic acid. Deletion of hamE resulted in sexual sterility and reductions in aflatoxin B1 production, however, asexual sporulation was not hindered. In A. fumigatus, via interactome data, we provided evidence of the existence of a pentameric pheromone module pathway, similar to what is observed in A. nidulans. The proteins of this pathway exhibited similar sub-cellular localisation patterns to those observed in A. flavus. HamE, MkkB and MpkB localise to the hyphal tips, suggesting that they form a trimeric complex. SteC interacts with SteD and this dimer associates with the trimeric complex in the cytoplasm to form a pentameric complex, allowing for kinase phosphorylation and translocation of MpkB into the nucleus. Deletion of any of the five pheromone module components results in dramatic reductions in asexual sporulation, vegetative growth rate and production of various secondary metabolites, particularly gliotoxin. Each mutant also exhibited increased sensitivity to both cell wall and oxidative stress agents. Overall, this thesis provides a comprehensive analysis of the molecular roles of the pheromone module in Aspergillus species. These data suggest that this pathway is a conserved mechanism of signal transduction that is required for the regulation of fungal development and secondary metabolism. Findings from this thesis may contribute to the development of strategies which could involve targeting the components of the pheromone module in order to help prevent crop spoilage and infections caused by Aspergillus species.

Item Type:	Thesis (PhD)
Keywords:	Conserved MAP Kinase Pathway; Regulation of Development and Secondary Metabolism; Aspergillus Species;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	13900
Depositing User:	IR eTheses
Date Deposited:	26 Jan 2021 15:37
URI:
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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