The RAM signaling pathway links morphology, thermotolerance, and CO2 tolerance in the global fungal pathogen Cryptococcus neoformans

  1. Benjamin J Chadwick
  2. Tuyetnhu Pham
  3. Xiaofeng Xie
  4. Laura C Ristow
  5. Damian J Krysan
  6. Xiaorong Lin  Is a corresponding author
  1. University of Georgia, United States
  2. University of Iowa, United States

Abstract

The environmental pathogen Cryptococcus neoformans claims over 180,000 lives each year. Survival of this basidiomycete at host CO2 concentrations has only recently been considered an important virulence trait. Through screening gene knockout libraries constructed in a CO2-tolerant clinical strain, we found mutations leading to CO2 sensitivity are enriched in pathways activated by heat stress, including calcineurin, Ras1-Cdc24, cell wall integrity, and Regulator of Ace2 and Morphogenesis (RAM). Overexpression of Cbk1, the conserved terminal kinase of the RAM pathway, partially restored defects of these mutants at host CO2 or temperature levels. In ascomycetes such as Saccharomyces cerevisiae and Candida albicans, transcription factor Ace2 is an important target of Cbk1, activating genes responsible for cell separation. However, no Ace2 homolog or any downstream component of the RAM pathway has been identified in basidiomycetes. Through in vitro evolution and comparative genomics, we characterized mutations in suppressors of cbk1D in C. neoformans that partially rescued defects in CO2 tolerance, thermotolerance, and morphology. One suppressor is the RNA translation repressor Ssd1, which is highly conserved in ascomycetes and basidiomycetes. The other is a novel ribonuclease domain-containing protein, here named PSC1, which is present in basidiomycetes and humans but surprisingly absent in most ascomycetes. Loss of Ssd1 in cbk1D partially restored cryptococcal ability to survive and amplify in the inhalation and intravenous murine models of cryptococcosis. Our discoveries highlight the overlapping regulation of CO2 tolerance and thermotolerance, the essential role of the RAM pathway in cryptococcal adaptation to the host condition, and the potential importance of post-transcriptional control of virulence traits in this global pathogen.

Data availability

Sequences generated from this research has been deposited to the Sequence Read Archive (SRA) under project accession number: PRJNA791949.

The following data sets were generated

Article and author information

Author details

  1. Benjamin J Chadwick

    Department of Plant Biology, University of Georgia, Athens, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8244-6190
  2. Tuyetnhu Pham

    Department of Plant Biology, University of Georgia, Athens, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Xiaofeng Xie

    Department of Microbiology, University of Georgia, Athens, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Laura C Ristow

    Department of Pediatrics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Damian J Krysan

    Department of Pediatrics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Xiaorong Lin

    Department of Microbiology, University of Georgia, Athens, United States
    For correspondence
    Xiaorong.Lin@uga.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3390-8387

Funding

National Institutes of Health (R01AI147541)

  • Damian J Krysan
  • Xiaorong Lin

National Institutes of Health (R01AI140719)

  • Xiaorong Lin

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed according to the guidelines of NIH and the University of Georgia Institutional Animal Care and Use Committee (IACUC). The animal models and procedures used have been approved by the IACUC (AUP protocol numbers: A2017 08-023 and A2020 06-015).

Reviewing Editor

  1. Arturo Casadevall, Johns Hopkins Bloomberg School of Public Health, United States

Publication history

  1. Received: August 9, 2022
  2. Accepted: November 22, 2022
  3. Accepted Manuscript published: November 23, 2022 (version 1)

Copyright

© 2022, Chadwick et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 55
    Page views
  • 26
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Benjamin J Chadwick
  2. Tuyetnhu Pham
  3. Xiaofeng Xie
  4. Laura C Ristow
  5. Damian J Krysan
  6. Xiaorong Lin
(2022)
The RAM signaling pathway links morphology, thermotolerance, and CO2 tolerance in the global fungal pathogen Cryptococcus neoformans
eLife 11:e82563.
https://doi.org/10.7554/eLife.82563

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Joseph V Geisberg, Zarmik Moqtaderi ... Kevin Struhl
    Research Advance

    Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' UTRs. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at the nucleotide level within clusters, but not between them. Pol II occupancy increases just downstream of the most speed-sensitive poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that 1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, 2) poly(A) site clusters are linked to the local elongation rate and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, 3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and 4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

    1. Genetics and Genomics
    Amel Lamri, Jayneel Limbachia ... Sonia S Anand
    Research Article Updated

    South Asian women are at increased risk of developing gestational diabetes mellitus (GDM). Few studies have investigated the genetic contributions to GDM risk. We investigated the association of a type 2 diabetes (T2D) polygenic risk score (PRS), on its own, and with GDM risk factors, on GDM-related traits using data from two birth cohorts in which South Asian women were enrolled during pregnancy. 837 and 4372 pregnant South Asian women from the SouTh Asian BiRth CohorT (START) and Born in Bradford (BiB) cohort studies underwent a 75-g glucose tolerance test. PRSs were derived using genome-wide association study results from an independent multi-ethnic study (~18% South Asians). Associations with fasting plasma glucose (FPG); 2 hr post-load glucose (2hG); area under the curve glucose; and GDM were tested using linear and logistic regressions. The population attributable fraction (PAF) of the PRS was calculated. Every 1 SD increase in the PRS was associated with a 0.085 mmol/L increase in FPG ([95% confidence interval, CI=0.07–0.10], p=2.85×10−20); 0.21 mmol/L increase in 2hG ([95% CI=0.16–0.26], p=5.49×10−16); and a 45% increase in the risk of GDM ([95% CI=32–60%], p=2.27×10−14), independent of parental history of diabetes and other GDM risk factors. PRS tertile 3 accounted for 12.5% of the population’s GDM alone, and 21.7% when combined with family history. A few weak PRS and GDM risk factors interactions modulating FPG and GDM were observed. Taken together, these results show that a T2D PRS and family history of diabetes are strongly and independently associated with multiple GDM-related traits in women of South Asian descent, an effect that could be modulated by other environmental factors.