Interaction of Cryptococcus neoformans Rim101 and protein kinase A regulates capsule

Abstract

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101 multiply sign in circle mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101 multiply sign in circle strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101 multiply sign in circle mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions.

Figure 1. C. neoformans Rim101 is required for capsule attachment.

A. rim101Δ mutants have a capsule defect. Cells were incubated in capsule inducing conditions for 3 days. Capsule was assessed by staining with India ink and visualizing the zone of exclusion at 63× magnification (scale bar = 10 m). B. The rim101Δ mutant sheds equivalent capsule to wild-type. Electrophoretic mobility and quantity of shed polysaccharide was assessed by a blotting technique of culture medium filtrate, using an anti-GXM antibody to probe for capsule as previously described . Cells were incubated in Dulbecco's modified Eagle's medium or low iron medium for 1 week before filtering. Arrow indicates direction of electrophoresis. C. Gfp-tagged Rim101 is functional. Cells were incubated in capsule inducing conditions for 3 days. Capsule was assessed by staining with India ink and visualizing the zone of exclusion at 63× magnification (scale bar = 10 m). D. Rim101-S773A does not complement capsule. Cells were incubated in capsule inducing conditions for 3 days. Capsule was assessed by staining with India ink and visualizing the zone of exclusion at 63× magnification (scale bar = 10 m).

Figure 2. Rim101 retains conserved phenotypes from other fungal species.

A. The rim101Δ mutant is sensitive to alkaline pH. Cells were incubated in buffered YNB, and growth was determined by cell counts after 72 hours. B. The rim101Δ mutant is sensitive to salt stress. 1×10⁵ cells from each strain were serially diluted (5-fold dilution) onto YPD plates containing 1.5M NaCl or 200 mM LiCl. The plates were incubated at 30°C for 3 days. Cells were plated onto YPD plates for 48 hours as a control.

Figure 3. Rim101 localization in mutant backgrounds.

Rim101 localization is dependent on PKA and Rim20. The pattern of Gfp-Rim101 localization in the indicated strains was visualized by DIC and fluorescent microscopy at 63× magnification and by confocal microscopy at 100× magnification.

Figure 4. Western blot analysis of Rim101 in rim101 and pka1Δ mutant backgrounds.

A. Rim101cleavage is dependent on PKA and Rim20. Immunoprecipitated Gfp-Rim101 from rim101Δ+ Gfp-rim101, rim101Δ + Gfp-rim101-S773A, pka1Δ + Gfp-rim10, rim20Δ + Gfp-rim101, and rim20Δ + Gfp-rim101-S773A strains was immunoblotted using anti-GFP antibody. B. Rim101 is cleaved after induction in capsule media. Gfp-Rim101 was immunoprecipitated from rim101Δ, rim101Δ + rim101-S773A, rim20Δ + Gfp-rim101, and rim20Δ + Gfp-rim101-S773A cell lysates after incubation in either YPD medium or the capsule-inducing medium DMEM at 30°C to mid-log phase. Samples were run on Bis-Tris gels and immunoblotted using an anti-GFP antibody.

Figure 5. Rim101 in low iron media.

A. Rim101 is required for growth in low-iron medium. Strains were incubated in low-iron medium and growth was quantified by monitoring the absorbance of the culture at 600 nm. B. The 5′-untranslated regions of C. neoformans genes involved in iron regulation were evaluated for the presence of putative Rim101 binding sites (GCCAAG or CCAAGAA).

Figure 6. Virulence analysis of the rim101Δ mutant strain.

A. rim101Δ mutant strains are hypervirulent in the murine inhalation model of cryptococcosis. AJc/r mice were inoculated intranasally with 5×10⁵cryptococcal cells and monitored for survival. B. rim101Δ mutant cells survive better than wild-type within macrophages. The rim101Δ mutant and isogenic wild-type strains were co-incubated with J744.1 macrophage-like cells previously activated by INF-gamma and LPS. Extracellular yeast cells were removed after one hour of co-incubation. After 24 hours of co-culture, the macrophages were lysed with SDS, and surviving yeast cells were quantitatively cultured. To precisely control for the number of added cells, the colony-forming units from each strain were normalized to that of wild-type cells. Data points represent the average of triplicate samples +/− standard error.