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The fungal pathogen Zymoseptoria tritici causes the most economically important disease of wheat in Europe. Despite recent advances in our understanding of its molecular host-pathogen interaction, fundamental questions remain about the cellular processes underlying plant colonisation by this fungus. The work presented in this thesis uses both reverse and forward genetic techniques to further understand the molecular determinants of Z. tritici virulence. To address questions about the source of nutrients utilised by Z. tritici to support symptomless colonisation of the leaf, this thesis explores cellular pathways utilised by other plant pathogenic fungi to access stored macromolecules. While autophagy is crucial for the infection-related development of many fungal plant pathogens, this study reveals that autophagy is dispensable for Z. tritici pathogenicity, and points towards a potential autophagy-independent function of ZtATG8 in virulence. The mitochondrial fatty acid β-oxidation pathway was however found to support the switch to hyphal growth on the leaf surface, providing strong evidence that catabolism of stored lipids is required for early host invasion by Z. tritici. Forward genetic investigation identified enzymes within the cell wall integrity (CWI) and cyclic adenosine monophosphate (cAMP) signalling pathways as playing a key role in Z. tritici virulence. In planta transcriptomic analysis revealed that the CWI pathway regulates the expression of infection-related secreted proteins, including the characterised LysM effectors required for host defence evasion, suggesting that Z. tritici may co-regulate virulence gene expression with the response to cell wall perturbation. Findings presented here also suggest that cAMP signalling regulates transcription during the switch to necrotrophic growth, providing insights into the elusive mechanisms controlling this infection cycle transition. Finally, genomic and transcriptomic analysis of a spontaneous Z. tritici mutant revealed the potential function of the light responsive transcription factor white collar 1 in controlling Z. tritici morphological development and infection. These novel findings advance our understanding of the cellular pathways contributing to Z. tritici infection and inform the development of future strategies to control this devastating pathogen.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease with the potential to cause chronic and life-threatening outcomes. This parasite spends its life alternating between a mammalian host and an arthropod vector, where it differentiates into four main developmental stages. How T. cruzi senses environmental changes that trigger cellular responses necessary for parasite survival has remained largely an enigma. Cyclic AMP is a universal second messenger that has been linked to several key cellular processes in trypanosomes, as part of a PKA-independent signaling pathway. Our research shows that T. cruzi cyclic AMP response protein 3 (TcCARP3) colocalizes with adenylate cyclase 1 (TcAC1) throughout the parasite life cycle and is involved in cAMP signaling by directly interacting with several adenylate cyclases and modulating the intracellular content of cAMP. By generation of TcCARP3 knockout, add back, and overexpression cell lines we demonstrated that modulation of protein expression affects the parasite’s ability to grow, differentiate, respond to osmotic stress, invade and replicate within mammalian cells, and colonize the hindgut of the triatomine vector. Our results unveil a key role for TcCARP3 in environmental sensing and life cycle progression of T. cruzi.

Cholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host quality control mechanism called ER-associated degradation (ERAD) that facilitates the translocation of misfolded proteins into the cytosol for degradation. Cytosolic CTA1, however, escapes this fate and is then free to activate its target, heterotrimeric G-protein subunit alpha (Gsα), leading to adenlyate cyclase (AC) hyperactivation and increased cAMP concentrations. This causes the secretion of chloride ions and water into the intestinal lumen. The result is severe diarrhea and dehydration which are the major symptoms of cholera. CTA1’s ability to exploit vesicle-mediated transport and ERAD for cytosolic entry demonstrates a potential link between cholera intoxication and a separate quality control mechanism called the unfolded protein response (UPR), which up-regulates vesicle-mediated transport and ERAD during ER stress. Other toxins in the same family such as ricin and Shiga toxin were shown to regulate the UPR, resulting in enhanced intoxication. Here, we show UPR activation by CT, which coincides with a marked increase in cytosolic CTA1 after 4 hours of toxin exposure. Drug induced-UPR activation also increases CTA1 delivery to the cytosol and increases cAMP concentrations during intoxication. We investigated whether CT stimulated UPR activation through Gsα or AC. Chemical activation of Gsα induced the UPR and increased CTA1 delivery to the cytosol. However, AC activation did iv not increase cytosolic CTA1 nor did it activate the UPR. These data provide further insight into the molecular mechanisms that cause cholera intoxication and suggest a novel role for Gsα during intoxication, which is UPR activation via an AC-independent mechanism

We studied the hypothesis that disturbances in 5-HT_{6} receptor function in the temporal cortex may contribute to clinical symptoms of Alzheimer's disease (AD). 5-HT_{6} density and 5-HT levels were significantly decreased in a cohort of AD patients prospectively assessed for cognitive/behavioral symptoms. cAMP formation after stimulation with the selective 5-HT_{6} receptor agonist E-6801 was significantly lower (p

Glucose detection in yeast occurs via a cAMP signaling pathway that is similar to that of other signaling pathways in humans. The presence of glucose in the environment ultimately represses, as a result of cAMP signaling, the transcription of the gene fbp1. Adenylate cyclase is known to convert ATP to cAMP, and is thus a central protein in the propagation of the signal. Mutant forms of the adenylate cyclase gene (git2) have been found by the inability for the organism to repress fbp1 transcription in the presence of glucose. In this study, two questions were under investigation. The first was focused on the ability of the mutations to affect the dimerization of the catalytic domain. The second investigated multiple protein-protein interactions in the leucine rich-repeat (LRR) domain of adenylate cyclase. Both domains contain mutations that confer an activation defect, and they are thus are thought to have a relationship.

The mechanism by which nicotinamide adenine dinucleotide (NAD) stimulates the activity of adenylate cyclase (AC) in canine plasma membrane has been studied. Using [3 2P]-NAD, the activation by NAD was correlated with the radiolabeling of the stimulatory guanosine triphosphate (GTP) binding protein Gsa. Further characterization demonstrated that the modification occurred only in the presence of G-protein activators and that arginine residue(s) were modified by ADP-ribose by the action of a mono-ADP-ribosyltransferase. Inhibitors of the transferase blocked both the modification of Gsa and the activation of AC. Collectively, these studies suggest that ADP-ribosylation of Gsa by an endogenous mono-ADP-ribosyltransferase may regulate cardiac AC.