Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation

ACKNOWLEDGMENTS We acknowledge Jennifer Lodge, Woei Lam, and Rajendra Upadhya for developing and sharing the chitin and chitosan MTBH assay. We thank Todd Brennan of Duke University for providing MyD88-deficient mice. We acknowledge Neil Gow for providing access to the Dionex HPAEC-PAD instrumentation. We also acknowledge Connie Nichols for critical reading of the manuscript. These experiments were supported by an NIH grant to J.A.A. and F.L.W., Jr. (R01 AI074677). C.M.L.W. was supported by a fellowship provided through the Army Research Office of the Department of Defense (no. W911NF-11-1-0136 f) (F.L.W., Jr.). J.W., L.W., and C.M. were supported by the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377) and the MRC, Centre for Medical Mycology (MR/N006364/1). FUNDING INFORMATION MRC Centre for Medical MycologyMR/N006364/1 Carol A. Munro HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) https://doi.org/10.13039/100000060R01 AI074677J. Andrew Alspaugh Wellcome https://doi.org/10.13039/100010269097377 Carol A. Munro DOD | United States Army | RDECOM | Army Research Office (ARO) https://doi.org/10.13039/100000183W911NF-11-1-0136 f Chrissy M. Leopold WagerPeer reviewedPublisher PD

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition

Acknowledgments We acknowledge Jeanette Wagener and Louise Walker for performing the HPAEC-PAD analysis and Neil Gow for providing access to the Dionex HPAEC-PAD instrumentation. We thank Mike Cook and the Duke University Cancer Center Flow Cytometry Shared Resource for assistance with the flow cytometry. We also acknowledge Michelle Plue and the Duke University Shared Materials Institute Facility for performing the transmission electron microscopy. We thank Marcel Wu¨thrich for providing the MyD88-/-and TLR2/4-/- mice, and Mari Shinohara and Elizabeth Deerhake for providing the Dectin-1-/- mice. Funding: These experiments were supported by a National Institutes of Health grant awarded to JAA and FLW, Jr. (R01 AI074677, https://grants.nih.gov/grants/oer.html). CM and colleagues Jeanette Wagener, Louise Walker, Neil Gow were supported by the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377, https://wellcome.ac.uk), Wellcome Trust Senior Investigator Award (101873) and the MRC Centre for Medical Mycology (MR/N006364/1, https://www.abdn.ac.uk/cmm/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Bacterial interactions with fungi in the vaginal tract take on many forms.

Metabolic products, surface proteins, and isolated bacterial components can all impact fungal growth and physiology at the epithelial surface. The vaginal microbiome similarly affects host epithelial biology and immune responses to commensal and pathogenic organisms. In combination, the activities of bacteria and fungi in this environment contribute to the pathology of vaginal infections. Figure created using BioRender.com.</p

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

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

The <i>rra1Δ</i> mutant is phenotypically identical to other Rim pathway mutants.

<p>(A) <i>rra1Δ</i> insertional mutant has pH 8 and 1.5 M NaCl growth defects that are rescued by <i>GFP-RIM101T</i> 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 <i>rra1Δ</i> mutant has a growth defect pH 8 and 1.5 M NaCl. (C) The <i>rra1Δ</i> 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 <i>rra1Δ</i> 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 <i>rra1Δ</i> mutant. GFP-Rim101 was assessed after 5 hr incubation in pH 8 SC McIlvaine’s buffer.</p

Rim101 proteolysis and nuclear localization are dependent on pH.

<p>(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) <i>rim101Δ</i> 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.</p

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

<p>A model of the canonical Rim pathway elucidated in ascomycete fungi [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005159#pgen.1005159.ref021" target="_blank">21</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005159#pgen.1005159.ref024" target="_blank">24</a>].</p

Effects of Rim pathway mutants on virulence.

<p>(A) <i>rim13Δ</i>, <i>rim23Δ</i>, and <i>rra1Δ</i> 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 <i>C</i>. <i>neoformans</i> cells.</p

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

<p>(A) The <i>C</i>. <i>neoformans RIM13</i> and <i>RIM23</i> 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 <i>rim13Δ</i> and <i>rim23Δ</i> mutants have a <i>rim101Δ-</i>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 <i>rim13Δ</i>, <i>rim20Δ</i>, and <i>rim23Δ</i> 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.</p

Strain list.

<p>Strain list.</p