Characterization of novel components of the environmental pH-sensing complex in Cryptococcus neoformans

Pathogenic microorganisms must adapt to changes in their surroundings, including alterations in pH, to survive the shift from the external environment to that of the infected host. In the basidiomycete fungal pathogen Cryptococcus neoformans, these pH changes are primarily sensed by the fungal-specific, alkaline pH-sensing Rim/Pal pathway. The C. neoformans Rim pathway has diverged significantly from the pathway described in ascomycete fungi. We recently identified the C. neoformans Rra1 putative pH sensor, which activates the Rim pathway in response to elevated pH. In this study, we probed the function of Rra1, by analyzing its cellular localization and performing protein co-immunoprecipitation experiments to identify potential Rra1 interactors. We found that Rra1 does not strongly colocalize or interact with immediate downstream Rim pathway components. However, these studies identified a novel Rra1 interactor, the previously uncharacterized C. neoformans Nucleosome Assembly Protein 1, Nap1, which is required for Rim pathway activation. Nap1 specifically binds to the C-terminal tail of the Rra1 sensor, likely promoting Rra1 protein stability. This novel function of Nap1 is conserved in closely-related fungi that contain Rra1 orthologs, but not in the more distantly-related ascomycete fungus, Saccharomyces cerevisiae. Therefore, these studies demonstrate novel means by which distinct microbial phyla have adapted sophisticated yet distinct means of rapidly adapting to environmental signals such as alterations in pH. As any cell transitions from one environment to another, it must adapt to variations in its extracellular conditions. Specifically, microbial pathogens must rapidly sense and adapt to the many changes in environment that are encountered upon entering a host. For example, a human host might present many extreme stresses relating to the external environment for a pathogen, including an elevated temperature, alterations in pH, limited nutrient availability, and other stresses such as those associated with an activated immune response. The pathogen must respond to these many stressful conditions in order to survive in the host. One of the most important ways in which fungi sense their external environment is through the Rim/Pal pH response pathway. This fungalspecific pathway is involved in cellular signaling in response to alkaline pH, ultimately regulating gene expression in order to adapt to this environmental stressor. This pathway is required for survival at elevated pH across many fungal species (1–3). First identified in Saccharomyces cerevisiae and Aspergillus nidulans, the Rim/Pal pathway, respectively, was further explored for roles in fungal pathogens, such as Candida albicans, Cryptococcus neoformans, and A. fumigatus (4–8). Cryptococcus neoformans Nap1 and pH sensing 2 In these pathogenic organisms, this alkaline pHactivated signaling pathway controls the expression of many genes required for full virulence (1, 9, 10). Indeed, many pathogenic species seem to have coopted this pathway for use as a sensor of host conditions. For example, as it transitions to the neutral to alkaline pH of the host, the human fungal pathogen C. albicans requires its Rim signaling pathway to activate genes involved in the yeasthyphal transition, a process required for tissue invasion (2, 6). This alkaline response pathway is also required for tissue invasion and pathogenesis of the filamentous opportunistic fungus, A. fumigatus (8). The yeast-like fungus C. neoformans is a neuropathogen in immunocompromised hosts, and the RIM101 transcription factor gene is one of the most highly induced genes in the setting of cryptococcal infection (11). Interestingly, C. neoformans Rim pathway signaling is required for full expression of the most important cell feature associated with Cryptococcus pathogenesis, the polysaccharide capsule (7). Together, these observations underscore the importance for microbial pathogens to correctly interpret host signals, such as elevated pH, and translate these signals to adaptive microbial responses. The Rim pathway has been most extensively studied in fungi from the Ascomycete phylum, including S. cerevisiae, C. albicans, A. nidulans, and A. fumigatus (12–14). In more distantly related fungi, such as the basidiomycete fungus C. neoformans, many of the Rim pathway signaling elements are conserved, including the Endosomal Sorting Complex Required for Transport (ESCRT) machinery which acts a scaffold for Rim pathway proteins during pathway activation, the Rim proteolysis complex (Rim13, Rim20, Rim23), and the Rim101 transcription factor (7, 15). These Rim pathway effectors not only represent homologs by sequence, but these proteins also function within the Rim pathway to transduce signals in response to alkaline pH. However, many orthologs of the upstream signaling components of the Rim pathway, including the pH sensor, are not present in the genomes of most basidiomycetes as assessed by direct sequence similarity. Previously, we identified the Rra1 protein as a putative pH sensor required for activation of the C. neoformans Rim pathway (15). Like the canonical Rim pathway pH sensor, Rra1 has seven predicted transmembrane domains and functions upstream of the Rim101 processing complex, including the ESCRT and Rim proteolysis complexes. Additionally, though no basidiomycetous fungi appear to have homologs of the Rim21 pH sensor, many—such as Cryptococcus gattii, Ustilago maydis, and Tremella mesenterica—have Rra1 homologs (15). While Rra1 may share structural similarities with the canonical Rim pathway pH sensor, it is unclear whether it activates downstream Rim pathway components in a similar manner. In ascomycetes, the Rim21/PalH pH sensor directly interacts with downstream Rim pathway components to induce Rim101/PacC processing complex assembly at the plasma membrane (13, 14, 16, 17). Additionally, we functionally placed the C. neoformans Rra1 protein within the Rim pathway as the most upstream component yet identified. However, efforts to identify homologs of other upstream effectors of the Rim pathway in C. neoformans through purely genetic means have not yet revealed other potential components of the Rim membrane sensing complex, including the Rim8/PalF arrestinlike protein that bridges the interaction between the pH sensor and the Rim proteolysis complex in ascomycetes (15, 18, 19). Therefore, it is still unclear how Rra1 functions in the C. neoformans Rim pathway. In this work, we searched for novel upstream regulators of the Rim pathway in C. neoformans through the identification of specific interactors of the Rra1 pH sensor. We employed proteomic methods to identify potential Rra1 interactors, subsequently using genetic techniques to determine whether these potential interactors regulated the activation of the Rim pathway in C. neoformans. We functionally placed one of these identified interactors, Nucleosome Assembly Protein 1 (Nap1), in the Rim pathway as a Rra1 signaling partner. Based on our evidence, we propose that C. neoformans Nap1 displays a novel, basidiomycete-specific function in Rim pathway activation through its interaction with the Rra1 pH sensor. Results Rra1, an upstream regulator of the Rim pathway, localizes to punctate structures at the plasma membrane Cryptococcus neoformans Nap1 and pH sensing 3 We previously identified a seventransmembrane domain-containing protein, Rra1 (Required for Rim101 Activation 1), that acts upstream of the C. neoformans Rim pathway proteolysis complex (15). To determine if this Rra1 protein might serve as a sensor of extracellular pH, we created a Rra1-GFP fusion protein to assess its subcellular localization. This pHIS3-RRA1-GFP fusion protein construct consists of the RRA1 gene fused to GFP under control of the constitutivelyactive Histone H3 promoter. The pHIS3-RRA1GFP construct was expressed in the rra1Δ mutant and shown to be functional, effectively rescuing the phenotypic defects demonstrated by the rra1Δ mutant strain, such as deficient growth on YPD pH8 and YPD + 1.5 M NaCl (Fig. S1A). When incubated at pH 4 or pH 8, the Rra1-GFP protein localized diffusely within endomembrane-like structures. This endomembrane pattern of fluorescence resembles perinuclear ER localization, which can be visualized by the ER marker Sec63 (Fig. 1A, B). Strikingly, the Rra1GFP protein localized most intensely in puncta on the cell surface, particularly at pH 4 (Fig. 1A). These puncta are present near the plasma membrane regardless of pH. However, upon pathway activation at pH 8, these cell surface puncta appear to decrease in number and, at times, to move to an intracellular location rather than being confined mostly to the cell surface as in pathway-inactivating conditions. These data suggest that Rra1-GFP is located and is potentially functioning at the cell surface. Moreover, the Rra1GFP fusion protein is likely being endocytosed upon pathway activation. The C. neoformans Rim pathway proteolysis complex consists of the ESCRT machinery (as a membrane-bound scaffolding component) and the Rim pathway-specific proteolysis complex components Rim13, Rim20, and Rim23 (15). To probe the spatial relationship between the Rra1 protein and other Rim pathway components, we expressed the Rra1-GFP construct in a strain expressing the ESCRT machinery component Vps23 tagged with mCherry (pHIS3mCherry-VPS23. When visualized using Z-stacked microscopic images, mCherry-Vps23 localizes to large, rare punctate structures within the cytosol, with most cells displaying only one of these structures (Fig. 1C). This strikingly focal, punctate pattern of mCherry-Vps23 fluorescence at pH 4 became more diffuse after a shift to pH 8, but it retained its cytosolic location (Fig 1C). Although the Rra1-GFP signal also included distinct punctate structures, the Rra1-GFP puncta were present at the cell surface as opposed to the cytosol. Additionally, we did not observe colocaliz