Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in <i>Cryptococcus</i>

<div><p>Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus <i>Cryptococcus neoformans</i> to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in <i>C</i>. <i>neoformans</i>. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.</p></div

Characterization of the sexual development of deletion mutants for spore-enriched proteins.

<p>(A) <i>rsc9Δ</i> strains show defects in mating, while <i>isp1Δ</i> and <i>bch1Δ</i> strains do not (top panels). Strains were mixed, incubated on V8 plates for 24h at 25°C, scraped up and visualized. Fusants (indicated by arrows) and yeast were counted. The number of fusants as a portion of total cell number is represented graphically for each strain (bottom panel). Data represent four individual experiments and are shown as mean ± standard deviation (SD). Scale bars, 10μm (100x magnification). (B) <i>rsc9Δ</i>, <i>isp1Δ</i>, and <i>bch1Δ</i> crosses showed much less robust filamentation 24h after the start of sexual development. Panels show the periphery of a spot of a cross on V8 plate for 24h at 25°C. Scale bars, 50μm (200x magnification). (C) <i>ddi1Δ</i>, <i>dst1Δ</i>, and <i>top1Δ</i> strains showed defects in spore formation. Both wild type and mutant crosses showed robust filamentation after 5 days on V8 plate (upper panels), but only wild type produced chains of spores (lower panels; arrows indicate basidia and triangles indicate spore chains). Mutants produced basidia without spore chains. Scale bars, 50μm (200x magnification) for upper panels and 10μm for lower panels (400x magnification). Spore isolations using density gradient centrifugation yielded 1%±1%, 2%±1%, and 0%±0% spores relative to wild type crosses from <i>ddi1Δ</i>, <i>dst1Δ</i>, and <i>top1Δ</i> crosses, respectively. (D) Quantified spore yield from density gradient purifications of mutant strains relative to wild type strains. <i>emc3Δ</i> and <i>gre202Δ</i> crosses yielded reproducible decreases in spore yield (approx. 2–4 fold) relative to wild type strains, whereas <i>isp2Δ</i> crosses produced more spores (approx. 1.5-fold higher) relative to wild type strains. Data represent number (n) of independent experiments and are shown as mean ± SD.</p

Yeast growth on YPD solid medium at 30°C.

<p>Yeast of the same starting concentration were spotted at 10-fold serial dilutions and grown for 3 days at 30°C. Multiple independent mutant strains were tested and representative growth for each mutant is shown.</p

<i>isp2Δ</i> spores show a delay in germination.

<p>(A) <i>isp2Δ</i> spores have a germination defect on solid YPD medium. Colonies of wild type and <i>isp2Δ</i> strains after growth at room temperature for 63h (germination) and 51h (vegetative growth). Scale bars, 100μm (5x magnification). (B) Quantification of colony sizes using ImageJ. The average colony size of <i>isp2Δ</i> spores was only 20.5% of wild type spores, whereas the average colony size of <i>isp2Δ</i> yeast was 45.7% of wild type yeast. The difference in size between colonies from yeast growth and spore germination for <i>isp2Δ</i> strains was significant (p = 7.1x10^-48) but not for the wild type strain (p = 7.8x10^-1). Data represent number (n) of independent experiments and are shown as a mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (C) Germination delay for <i>isp2Δ</i> spores in liquid YPD media. Optical density at a wavelength of 600nm (OD₆₀₀) was measured every 3min over 50h. The y-axis shows OD₆₀₀ and the x-axis shows time in hours (h). Plots are representative of three independent experiments. (D) Average time taken to double initial OD₆₀₀. Quantified doubling times were nearly identical for wild type and <i>isp2Δ</i> yeast (p = 0.38); however, <i>isp2Δ</i> spores took significantly longer than wild type to double the population (p = 1.2×10⁻¹⁰). Data represent number (n) of independent experiments and are shown as mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (E) Morphological changes during germination of <i>isp2Δ</i> and wild type spores. Spores were exposed to YPD liquid media to trigger germination at room temperature and photographed at 0h and 12h. Scale bars, 5μm (1000× magnification).</p

Implementation of Activated Ion Electron Transfer Dissociation on a Quadrupole-Orbitrap-Linear Ion Trap Hybrid Mass Spectrometer

Using concurrent IR photoactivation during electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), significantly increases dissociation efficiency resulting in improved overall performance. Here we describe the first implementation of AI-ETD on a quadrupole-Orbitrap-quadrupole linear ion trap (QLT) hybrid MS system (Orbitrap Fusion Lumos) and demonstrate the substantial benefits it offers for peptide characterization. First, we show that AI-ETD can be implemented in a straightforward manner by fastening the laser and guiding optics to the instrument chassis itself, making alignment with the trapping volume of the QLT simple and robust. We then characterize the performance of AI-ETD using standard peptides in addition to a complex mixtures of tryptic peptides using LC–MS/MS, showing not only that AI-ETD can nearly double the identifications achieved with ETD alone but also that it outperforms the other available supplemental activation methods (ETcaD and EThcD). Finally, we introduce a new activation scheme called AI-ETD+ that combines AI-ETD in the high pressure cell of the QLT with a short infrared multiphoton dissociation (IRMPD) activation in the low-pressure cell. This reaction scheme introduces no addition time to the scan duty cycle but generates MS/MS spectra rich in b/y-type and c/z^•-type product ions. The extensive generation of fragment ions in AI-ETD+ substantially increases peptide sequence coverage while also improving peptide identifications over all other ETD methods, making it a valuable new tool for hybrid fragmentation approaches

Assessment of the proteomic data.

<p>(A) Venn diagram of spore proteins and yeast proteins identified. 2232 and 2192 proteins were identified from spores and yeast, respectively, with a majority of 1858 existing in both cell types. (B) Virtual two-dimensional gel diagrams of the predicted <i>C</i>. <i>neoformans</i> proteome (upper panel) and identified proteins in either yeast (middle panel) or spores (lower panel). Each dot represents a protein, with the x-axis showing isoelectric point (pI) and the y-axis as molecular weight (MW, Dalton).</p

Eighteen genes encoding spore-enriched proteins.

<p>^a. Genes encoding proteins with no obvious homologs were named <i>ISP</i> for <u>I</u>dentified <u>S</u>pore <u>P</u>rotein.</p><p>Eighteen genes encoding spore-enriched proteins.</p

Spores derived from wild type by <i>isp2Δ</i> crosses show wild type rates of germination.

<p>(A) Spores purified from WT × <i>isp2Δ</i> strains were grown in YPD and measured via optical density (OD₆₀₀) every 3min over 50h. The y-axis shows OD₆₀₀ and the x-axis shows time in hours (h). Plots are representative of three independent experiments. (B) Model of Isp2 activity during germination. Germination encompasses two stages: a morphological transition and growth initiation before the active replication of vegetative growth. Isp2 protein (black triangles) is present in mature spores from WT × WT crosses and persists during germination through the morphological transition to contribute to optimal growth initiation. In contrast, there is no Isp2 in spores from <i>isp2Δ</i> × <i>isp2Δ</i> crosses, and thus, a delay of ~2h during germination occurs, specifically during the growth initiation phase. Notably, spores from WT × <i>isp2Δ</i> crosses do not show a delay in germination and therefore contain Isp2 protein similar to wild type spores, regardless of genotype. Spores are shown as ovals with stalks, whereas yeast are shown as spheres. Large and small spheres together represent budding yeast. Isp2 protein is represented by black triangles.</p

Number of proteins identified in spores and yeast.

<p>^a. Proteins identified only in spores but not in yeast</p><p>^b. Proteins identified only in yeast but not in spores</p><p>^c. Proteins identified in at least one replicate of spores or yeast</p><p>Number of proteins identified in spores and yeast.</p

Spore-overrepresented and yeast- overrepresented proteins.

<p>Functional annotation clustering analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The underlined GO terms expand those identified in spore-enriched proteins (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005490#pgen.1005490.g002" target="_blank">Fig 2</a>).</p><p>Spore-overrepresented and yeast- overrepresented proteins.</p