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. 2022 Apr 27;10(2):e0030122.
doi: 10.1128/spectrum.00301-22. Epub 2022 Apr 12.

Gluconate Kinase Is Required for Gluconate Assimilation and Sporulation in Cryptococcus neoformans

Andrew J Jezewski  1 , Sarah R Beattie  1 , Katy M Alden  1 , Damian J Krysan  1   2
Affiliations

Gluconate Kinase Is Required for Gluconate Assimilation and Sporulation in Cryptococcus neoformans

Andrew J Jezewski et al. Microbiol Spectr. .

Abstract

Cryptococcus neoformans is an environmental yeast and an opportunistic human pathogen. The ability to cause disease depends on the ability to adapt to the human host. Previous studies implicated infectivity-related kinase 3 (IRK3, CNAG_03048) as required for establishing an infection. We genetically and biochemically characterized IRK3 as a gluconate kinase and propose the name GNK1. This metabolic enzyme utilizes gluconate to produce 6-phosphogluconate as part of the alternative oxidative phase of the pentose phosphate pathway (AOXPPP). The presence of GNK1 confirms that the AOXPPP is present and able to compensate for loss of the traditional OXPPP, providing an explanation for its nonessentiality. C. neoformans can utilize gluconate as an alternative carbon source in a GNK1-dependent manner. In our efforts to understand the role of GNK1 in host adaptation and virulence, we found that GNK1-deficient mutants have variable virulence and carbon dioxide tolerance across multiple strains, suggesting that second site mutations frequently interact with GNK1 deletion mutations. In our effort to isolate these genetic loci by backcrossing experiments, we discovered that GNK1-deficient strains are unable to sporulate. These data suggest that gluconate metabolism is critical for sporulation of C. neoformans. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that contributes to nearly 180,000 deaths annually. We characterized a gene named GNK1 that appears to interact with other genetic loci involved with the ability of C. neoformans to act as a pathogen. While these interacting genetic loci remain elusive, we discovered that GNK1 plays roles in both metabolism and mating/sporulation. Further interrogation of the mechanistic role for GNK1 in sexual reproduction may uncover a larger network of genes that are important for host adaptation and virulence.

Keywords: Cryptococcus neoformans; carbon dioxide; gluconate kinase; metabolism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
GNK1 functions in the alternative oxidative pentose phosphate pathway. Schematic highlights the differences between the tradition oxidative phase of the pentose phosphate pathway (blue) and the alternative oxidative phase of the pentose phosphate pathway (yellow) with their shared final step (green). Steps that produce reducing equivalents are indicated by their production of NADPH. The metabolic role for Gnk1 is indicated by the labeled illustration (pink).
FIG 2
FIG 2
C. neoformans utilizes gluconate as a carbon source in a GNK1-dependent manner. Spot dilutions of gluconate kinase deficient mutants grown on YNB media with either (A) 2% glucose or (B) 2% gluconate supplied as the carbon source. (C) Multiple independent isolates of GNK1 deletion mutants in the H99 background as well as a GNK1 deletion from a separate clinical isolate (C23) were incubated on YNB-gluconate. The plating involves a 1:10 dilution series from starting an overnight culture adjusted to an OD600 = 1. FGSC indicates strains obtained from the Bahn Laboratory’s deletion collection available from the Fungal Genetics Stock Center (8).
FIG 3
FIG 3
Biochemical characterization of C. neoformans Gnk1. (A) Coomassie-stained SDS-PAGE gel of heterologously expressed CnGnk1. (B) Kinetic assay showing gluconate-dependent enzyme activity. Michaelis-Menten kinetics of Gnk1p substrates gluconate (C) and ATP (D) with the other substrate at saturating concentrations. The Michaelis-Menten constants are apparent because of the bi-substrate nature of the reaction. Curves are representative of experimental duplicates each containing technical duplicates and plotted using GraphPad Prism. Confidence intervals of Michaelis Menten constants reported in text.
FIG 4
FIG 4
GNK1 deletion mutants do not have consistent effects on carbon dioxide tolerance. Spot dilutions of GNK1 deletion mutants from the fungal genetics stock center (FGSC) incubated on YNB with 2% glucose media with either ambient or 5% carbon dioxide (A). Multiple, independently generated isolates derived from H99 and a GNK1 deletion from a separate clinically derived strain (C23) grown on YNB with either ambient or 5% carbon dioxide (B). The plating involves a 1:10 dilution series from starting an overnight culture adjusted to an OD600 = 1.
FIG 5
FIG 5
GNK1 deletion mutants do not have consistent effects on virulence. Survival of A/J mice infected via the tail vein with the H99 reference strain (n = 10) or gnk1Δ FGSC 1 from the kinase deletion library (n = 4) (A). Survival of A/J mice infected via the pulmonary route with H99 (n = 10) or gnk1Δ FGSC 1 (n = 10) (B). Brain burden of gnk1Δ FGSC 1 in A/J mice on day 32 of a pulmonary inoculation or day 14 of a tail vein inoculation (C). Survival of A/J mice infected via the pulmonary route with indicated strains, n = 6 for all strains (D). Southern blot of DNA isolated from C. neoformans recovered from mice in panel E. (F) Survival of A/J mice infected via the pulmonary route with a clinical isolate C23 (n = 6) and the C23-derived gnk1Δ mutant (n = 6).
FIG 6
FIG 6
GNK1 deletion mutants have a sporulation defect. Representative images from H99a crossed with KN99α (A) or a gnk1Δ mutant crossed with H99a (B) 8 weeks postplating on V8 agar. Large red boxes are enlargements of the regions outlined in red. Red arrows highlight spore chains present in the H99a × KN99α crossing. The black arrows in B indicate the presence of basidia without spore chains.
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