The effect of Rsp5-mediated ubiquitination on the pathogenesis of the human fungal pathogen Cryptococcus neoformans

Abstract

Cellular responses to external stress allow microorganisms to adapt to a vast array of environmental conditions, including sites of host infection. The molecular mechanisms behind these responses are studied to gain insight into microbial pathogenesis, which could lead to new anti-microbial therapies. Although fungi are often overlooked as microbial pathogens of humans, they contribute significantly to morbidity and mortality as opportunistic pathogens. Cryptococcus neoformans is one such opportunistic fungal pathogen common among immunocompromised individuals, especially those living with HIV/AIDS. This yeast-like fungus is surrounded by a polysaccharide capsule, which is dynamically regulated in response to environmental stimuli, including those encountered during infection of a human host. Several of the well-studied signaling pathways in C. neoformans influence polysaccharide capsule synthesis through tight regulation of transcription and protein activation by post-translational modification. While some forms of post-translational modification, such as phosphorylation, are well-explored, ubiquitination is less well understood in this fungus. Ubiquitination is known for labeling spent and misfolded proteins for degradation, but more recent work also found non-degradation related functions for ubiquitination, including activation of signaling pathways. I hypothesized that ubiquitination will also regulate the stress response pathways necessary for the pathogenesis of C. neoformans, including reshaping the cell surface and capsule. A candidate ubiquitinating enzyme for these studies, the Rsp5 E3 ubiquitin ligase, contains a C2 protein domain that allows it to tether to the plasma membrane, placing it in close proximity to the cell surface synthesis enzymes to allow for their ubiquitination. This ubiquitin ligase belongs to the HECT class of ubiquitin ligases and, although this ubiquitin ligase is specific to fungi, it has some sequence homology with the mammalian Nedd4 family of ubiquitin ligases. This ligase family often interacts with target proteins for ubiquitination indirectly through adapter proteins, most commonly proteins with arrestin domains. Through these interactions, the Nedd4 family and related ubiquitin ligases are able to regulate many different cellular processes, including those important for immune responses to microbial pathogens. In Chapter 2 I explore a role for arrestin protein-mediated ubiquitination in stress response and pathogenesis in C. neoformans. In a previous study we identified four arrestin-like proteins in C. neoformans and found that one of these is required for efficient membrane synthesis, likely by directing interaction between fatty acid synthases and the Rsp5 E3 ubiquitin ligase. I further explored Cn Rsp5 function and determined that this single ubiquitin ligase is absolutely required for pathogenesis and survival in the presence of cellular stress. Additionally, I show that a second arrestin-like protein, Ali2, similarly facilitates interaction between Rsp5 and some of its protein targets. Of the four postulated C. neoformans arrestin-like proteins, Ali2 appears to contribute the most to C. neoformans pathogenesis, likely by directing Rsp5 to pathogenesis-related ubiquitination targets. A proteomics-based differential ubiquitination screen revealed that several known cell surface proteins are ubiquitinated by Rsp5 and a subset also requires Ali2 for their ubiquitination. Rsp5-mediated ubiquitination alters the stability and the localization of these proteins. A loss of Rsp5-mediated ubiquitination results in cell wall defects that increase susceptibility to external stresses. These findings support a model in which arrestin-like proteins guide Rsp5 to ubiquitinate specific target proteins, some of which are required for survival during stress. Many fungi grow optimally in acidic environments, and fungal pathogens, therefore, often rely on the activation of stress response pathways such as the Pal/Rim pathway to adapt to the neutral to slightly basic pH encountered when infecting mammals. While this pathway is conserved among fungi, the proteins required for pH sensing appear to have diverged between different fungal phyla. Cryptococcus neoformans, a basidiomycete, employs a pH sensing protein in its Rim pathway that is distinct but functionally analogous to related proteins in well-studied ascomycete fungal systems. Findings described in Chapter 2 suggest that protein ubiquitination mediated through the Rsp5 ubiquitin ligase to be required for adaptation of C. neoformans to mammalian host pH levels. In Chapter 3 I determined that Cn Rsp5 is specifically required for Rim pathway activation. Using an unbiased screen for proteins that are ubiquitinated by Rsp5 in acidic and alkaline pH, I identified a new component of the C. neoformans Rim pathway that is targeted by Rsp5 for ubiquitination and shares protein features with the ascomycete Rim8/PalF proteins. Rsp5-mediated ubiquitination facilitates protein interactions with Vps23, a downstream trafficking component of the Pal/Rim pathway. Therefore, I define adaptation to ambient pH as one component of the broad cellular roles of Rsp5-mediated ubiquitination, as I explore how protein ubiquitination affects cryptococcal cell physiology, virulence, and microbial interactions with the host. In Chapter 4 I sought to identify direct binding partners of Rsp5 that might include novel adapter proteins of this ligase. I also explored the potential of Rsp5 as a target of ubiquitin ligase inhibitors that might be used as antifungal agents to treat C. neoformans infections, which are further studied in ongoing work. My studies explored the importance of Rsp5-mediated ubiquitination in the regulation of signaling pathways important for the pathogenesis of C. neoformans, leading to a better understanding of the dynamic process of host infection and colonization.