The Cryptococcus neoformans alkaline response pathway: identification of a novel rim pathway activator

Abstract

The Rim101/PacC transcription factor acts in a fungal-specific signaling pathway responsible for sensing extracellular pH signals. First characterized in ascomycete fungi such as Aspergillus nidulans and Saccharomyces cerevisiae, the Rim/Pal pathway maintains conserved features among very distantly related fungi, where it coordinates cellular adaptation to alkaline pH signals and micronutrient deprivation. However, it also directs species-specific functions in fungal pathogens such as Cryptococcus neoformans, where it controls surface capsule expression. Moreover, disruption of the Rim pathway central transcription factor, Rim101, results in a strain that causes a hyper-inflammatory response in animal infection models. Using targeted gene deletions, we demonstrate that several genes encoding components of the classical Rim/Pal pathway are present in the C. neoformans genome. Many of these genes are in fact required for Rim101 activation, including members of the ESCRT complex (Vps23 and Snf7), ESCRT-interacting proteins (Rim20 and Rim23), and the predicted Rim13 protease. We demonstrate that in neutral/alkaline pH, Rim23 is recruited to punctate regions on the plasma membrane. This change in Rim23 localization requires upstream ESCRT complex components but does not require other Rim101 proteolysis components, such as Rim20 or Rim13. Using a forward genetics screen, we identified the RRA1 gene encoding a novel membrane protein that is also required for Rim101 protein activation and, like the ESCRT complex, is functionally upstream of Rim23-membrane localization. Homologs of RRA1 are present in other Cryptococcus species as well as other basidiomycetes, but closely related genes are not present in ascomycetes. These findings suggest that major branches of the fungal Kingdom developed different mechanisms to sense and respond to very elemental extracellular signals such as changing pH levels.

Fig 1. A model of the canonical Rim pathway elucidated in ascomycete fungi [21,24].

Fig 2. Rim101 proteolysis and nuclear localization are dependent on pH.

(A) GFP-Rim101 is proteolytically processed from 140 kDa to ~100 kDa in response to increasing pH. GFP-Rim101 was immunoprecipitated from wild-type cells after incubating for 5 hr at the indicated pH 8 (SC medium buffered with McIlvaine’s buffer). Protein processing was determined by western blotting using an α-GFP antibody. (B) GFP-Rim101 nuclear localization increases in response to increasing pH. Cells were cultured in the same way as in (A). GFP signal was assessed by epifluorescence microscopy. Nuclei were stained using Hoechst 33342 live nuclei stain. Scale bar = 5 μm. (C) GFP-Rim101 proteolysis is not induced by 1 M NaCl or 150 μM BPS. Cells were cultured in each indicated condition for 3 hr. GFP-Rim101 was analyzed by western blot. (D) GFP-Rim101 localization in response to pH 7 SC (McIlvaine’s) or pH 4 SC (McIlvaine’s) with 1 M NaCl or 150 μM BPS. Cell assessed epifluorescence microscopy after 30 min incubation in each condition. Scale bar = 5 μm (E) rim101Δ is not NaCl sensitive at pH 4. Strains spotted onto YPD, YPD 150mM HEPES pH 4 1.5 M NaCl, and YPD 1.5 M NaCl.

Fig 3. Role of Rim13 and Rim23 orthologs in Rim101-regulated phenotypes.

(A) The C. neoformans RIM13 and RIM23 orthologs are required for pH 8 and NaCl tolerance. 10-fold serial dilutions of the indicated strains were spotted onto YPD, YPD 150mM HEPES pH 8, YPD 1.5M NaCl and incubated at 30^°C for 48 hr -72 hr (B) The rim13Δ and rim23Δ mutants have a rim101Δ-like capsule defect. Cells were incubated in CO₂-independent media for 48hr at 37^°C. Capsule was visualized by counterstaining with India ink. (C) Rim101 proteolysis and localization are disrupted in rim13Δ, rim20Δ, and rim23Δ mutant strains. GFP-Rim101 was immunoprecipitated from each strain after 5 hr incubation in pH 7.4 YPD buffered with 150 mM HEPES. (D) GFP-Rim101 localization was assessed in the indicated strains after culturing for 5 hr in SC medium buffered with McIlvaine’s buffered to pH 8. Nuclei were stained with Hoechst 33342 live nuclei stain. Scale bar = 5 μm.

Fig 4. ESCRT complex proteins, Vps23 and Snf7, are required for Rim101 activation.

(A) snf7Δ and vps23Δ capsule defects are partially rescued by GFP-RIM101T expression. Strains cultured for 24 hr in tissue culture media. India ink used to visualize capsule. (B) GFP-RIM101T expression rescues the vps23Δ and snf7Δ growth defects on pH 8 but not 1.5 M NaCl. (C) GFP-Rim101 was immunoprecipitated from the indicated strains after 5 hr incubation in YPD with 150mM HEPES at pH 7.4. (D) GFP-Rim101 (full-length) nuclear localization is disrupted in the vps23Δ mutant. Localization was assessed after culturing for 5 hr in SC with McIlvaine’s buffer at pH 8. Nuclei were stained with Hoechst 33342 live nuclear stain. Scale bar = 5 μm.

Fig 5. The rra1Δ mutant is phenotypically identical to other Rim pathway mutants.

(A) rra1Δ insertional mutant has pH 8 and 1.5 M NaCl growth defects that are rescued by GFP-RIM101T expression. 10-fold serial dilutions were spotted onto YPD, YPD with 150 mM HEPES pH 8, and YPD with 1.5 M NaCl. (B) Independent rra1Δ mutant has a growth defect pH 8 and 1.5 M NaCl. (C) The rra1Δ strain has a capsule defect. Cells were cultured for 48 hr in CO₂-independent media at 37^°C to induce capsule. Capsule was visualized by India ink staining. Scale bar = 5 μm. (D) The rra1Δ mutation disrupts GFP-Rim101 proteolysis. GFP-Rim101 was immunoprecipitated from the indicated mutant strains after 5 hr incubation in YPD with 150 mM HEPES at pH 7.4. (E) GFP-Rim101 nuclear localization is disrupted in the rra1Δ mutant. GFP-Rim101 was assessed after 5 hr incubation in pH 8 SC McIlvaine’s buffer.

Fig 6. Rra1 is a membrane protein and is conserved through Basidiomycete fungi.

(A) C. neoformans Rra1 and its predicted basidiomycete orthologs contain 7–8 predicted transmembrane domains (closed boxes). C. neoformans CNAG_03488 (Rra1), C. gattii CGB_G5320C, Tremella mesenterica TREME_69388, Ustilago maydis um00299, Puccinia graminis PGTG_03106 predicted protein sequences are modeled. The TM domains were predicted using The TMPred TM prediction server [38] The % identity to the transmembrane region of C. neoformans Rra1 was determined by comparing 1–290 of the predicted Rra1 amino acid sequence to each of the predicted orthologs. (B) C. gattii rra1Δ and rim101Δ mutants are sensitive to pH 8. (C) Cg rra1Δ and rim101Δ have a capsule defect. Capsules visualized with India Ink after 24 hr incubation in tissue culture media. Scale bar = 5 μm.

Fig 7. Rim23-GFP forms plasma membrane-associated puncta under neutral/alkaline pH conditions.

(A) Rim23-GFP was visualized at pH 4 SC McIlvaine’s buffer and after 30 min after shift to pH 7 SC McIlvaine’s buffer. (B) The number of Rim23-GFP puncta increase with time after shifting from pH 4 to pH 7. Quantification Rim23-GFP puncta/cell at pH 4 (0 min) and after 10, 20, 30 min incubation at pH 7. (C) Rim23-GFP puncta are closely associated with the plasma membrane. Cells were stained with FM-464 after 1 hr incubation at pH 7. (D) 1 M NaCl and 150 μM BPS does not induce Rim23-GFP puncta formation. Cells images after 30 min incubation in indicated culture media. (E) Rim23-GFP puncta formation was disrupted by rra1Δ, vps23Δ, and snf7Δ mutants. Cells were imaged after 30 min incubation at pH 7. The number of puncta/cell was quantified for 62–100 cells/strain. All strains in this figure were cultured in SC McIlvaine’s buffer media. All scale bars = 5 μm.

Fig 8. Effects of Rim pathway mutants on virulence.

(A) rim13Δ, rim23Δ, and rra1Δ mutants are hypervirulent in a murine model of cryptococcosis. 8–10 A/Jcr female mice were intranasally inoculated with 1X10⁵ cryptococcal cells and monitored daily for survival. (B) Histopathological analysis revealed increased inflammatory cell infiltration in Rim-pathway mutants. Infected A/Jcr mouse lungs were harvested on day 7 post inoculation and H & E stain. Black arrows mark C. neoformans cells.

Fig 9. Model of C. neoformans Rim pathway.

Our data supports a model in which the Rim pathway is activated by neutral/alkaline pH. In this pathway, the newly discovered Rra1 membrane protein and the ESCRT complex, including Vps23 (ESCRT-I) likely Vps25 (ESCRT-II) and Snf7 (ESCRT-III), induce Rim23 puncta formation and activate the Rim101-proteolysis complex. The Rim13 protease cleaves the Rim101 transcription factor, inducing its nuclear localization. Active Rim101 then transcriptional changes necessary for adapting to alkaline pH.